CA3097428A1 - Method for differentiation of ocular cells and use thereof - Google Patents
Method for differentiation of ocular cells and use thereofInfo
- Publication number
- CA3097428A1 CA3097428A1 CA3097428A CA3097428A CA3097428A1 CA 3097428 A1 CA3097428 A1 CA 3097428A1 CA 3097428 A CA3097428 A CA 3097428A CA 3097428 A CA3097428 A CA 3097428A CA 3097428 A1 CA3097428 A1 CA 3097428A1
- Authority
- CA
- Canada
- Prior art keywords
- cells
- prp
- population
- express
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 214
- 230000004069 differentiation Effects 0.000 title claims description 83
- 210000004027 cell Anatomy 0.000 claims abstract description 873
- 108091008695 photoreceptors Proteins 0.000 claims abstract description 42
- 239000002243 precursor Substances 0.000 claims abstract description 27
- 239000002609 medium Substances 0.000 claims description 158
- 102000018210 Recoverin Human genes 0.000 claims description 131
- 108010076570 Recoverin Proteins 0.000 claims description 131
- 239000003112 inhibitor Substances 0.000 claims description 120
- 230000014509 gene expression Effects 0.000 claims description 118
- 101000854931 Homo sapiens Visual system homeobox 2 Proteins 0.000 claims description 114
- 102100020676 Visual system homeobox 2 Human genes 0.000 claims description 114
- 230000002207 retinal effect Effects 0.000 claims description 72
- 108010032788 PAX6 Transcription Factor Proteins 0.000 claims description 63
- 102100037506 Paired box protein Pax-6 Human genes 0.000 claims description 63
- 102100028854 Sushi domain-containing protein 2 Human genes 0.000 claims description 61
- 102100025818 Major prion protein Human genes 0.000 claims description 60
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 60
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 56
- 238000012258 culturing Methods 0.000 claims description 55
- 239000000203 mixture Substances 0.000 claims description 42
- 210000001164 retinal progenitor cell Anatomy 0.000 claims description 41
- 102000013814 Wnt Human genes 0.000 claims description 34
- 108050003627 Wnt Proteins 0.000 claims description 34
- 229940121773 Secretase inhibitor Drugs 0.000 claims description 29
- 239000011668 ascorbic acid Substances 0.000 claims description 29
- 229960005070 ascorbic acid Drugs 0.000 claims description 29
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 28
- 235000010323 ascorbic acid Nutrition 0.000 claims description 28
- 101000713575 Homo sapiens Tubulin beta-3 chain Proteins 0.000 claims description 26
- OGKYMFFYOWUTKV-UHFFFAOYSA-N N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide Chemical compound C1=NC=C2C(S(=O)(=O)NCCN)=CC=C(Cl)C2=C1 OGKYMFFYOWUTKV-UHFFFAOYSA-N 0.000 claims description 26
- 102100036790 Tubulin beta-3 chain Human genes 0.000 claims description 26
- -1 Ki67 Proteins 0.000 claims description 25
- 210000001020 neural plate Anatomy 0.000 claims description 24
- 239000007739 pm medium Substances 0.000 claims description 23
- 102100039820 Frizzled-4 Human genes 0.000 claims description 22
- 230000035800 maturation Effects 0.000 claims description 21
- 238000004519 manufacturing process Methods 0.000 claims description 20
- 101000620359 Homo sapiens Melanocyte protein PMEL Proteins 0.000 claims description 19
- 102100022430 Melanocyte protein PMEL Human genes 0.000 claims description 19
- 102100024964 Neural cell adhesion molecule L1 Human genes 0.000 claims description 19
- CDOVNWNANFFLFJ-UHFFFAOYSA-N 4-[6-[4-(1-piperazinyl)phenyl]-3-pyrazolo[1,5-a]pyrimidinyl]quinoline Chemical compound C1CNCCN1C1=CC=C(C2=CN3N=CC(=C3N=C2)C=2C3=CC=CC=C3N=CC=2)C=C1 CDOVNWNANFFLFJ-UHFFFAOYSA-N 0.000 claims description 18
- 102100040120 Prominin-1 Human genes 0.000 claims description 18
- 229940124647 MEK inhibitor Drugs 0.000 claims description 17
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 claims description 17
- 230000001537 neural effect Effects 0.000 claims description 17
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 16
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 claims description 16
- 102100023064 Nectin-1 Human genes 0.000 claims description 16
- 230000006698 induction Effects 0.000 claims description 16
- 102100032063 Neurogenic differentiation factor 1 Human genes 0.000 claims description 14
- 239000011570 nicotinamide Substances 0.000 claims description 14
- 229960003966 nicotinamide Drugs 0.000 claims description 14
- 235000005152 nicotinamide Nutrition 0.000 claims description 14
- 238000003908 quality control method Methods 0.000 claims description 13
- 239000000725 suspension Substances 0.000 claims description 13
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 claims description 12
- 102100025222 CD63 antigen Human genes 0.000 claims description 12
- 102100029087 Hepatocyte nuclear factor 6 Human genes 0.000 claims description 12
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 claims description 12
- 101000988619 Homo sapiens Hepatocyte nuclear factor 6 Proteins 0.000 claims description 12
- 101000974834 Homo sapiens Sodium/potassium-transporting ATPase subunit beta-3 Proteins 0.000 claims description 12
- 102100022792 Sodium/potassium-transporting ATPase subunit beta-3 Human genes 0.000 claims description 12
- 102100029362 Cone-rod homeobox protein Human genes 0.000 claims description 11
- 108010023082 activin A Proteins 0.000 claims description 11
- 230000001464 adherent effect Effects 0.000 claims description 11
- 102100024894 PR domain zinc finger protein 1 Human genes 0.000 claims description 10
- 108010009975 Positive Regulatory Domain I-Binding Factor 1 Proteins 0.000 claims description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 101000919370 Homo sapiens Cone-rod homeobox protein Proteins 0.000 claims description 9
- 108010050345 Microphthalmia-Associated Transcription Factor Proteins 0.000 claims description 9
- 102100030157 Microphthalmia-associated transcription factor Human genes 0.000 claims description 9
- 101150079937 NEUROD1 gene Proteins 0.000 claims description 9
- 108010012255 Neural Cell Adhesion Molecule L1 Proteins 0.000 claims description 9
- 108700020297 NeuroD Proteins 0.000 claims description 9
- 229940043378 cyclin-dependent kinase inhibitor Drugs 0.000 claims description 9
- 102100022794 Bestrophin-1 Human genes 0.000 claims description 8
- 101000903449 Homo sapiens Bestrophin-1 Proteins 0.000 claims description 8
- 101001078886 Homo sapiens Retinaldehyde-binding protein 1 Proteins 0.000 claims description 8
- 102100028001 Retinaldehyde-binding protein 1 Human genes 0.000 claims description 8
- 208000014674 injury Diseases 0.000 claims description 8
- 101100047651 Anopheles gambiae TRYP1 gene Proteins 0.000 claims description 7
- 102100030634 Homeobox protein OTX2 Human genes 0.000 claims description 7
- 101000584400 Homo sapiens Homeobox protein OTX2 Proteins 0.000 claims description 7
- 101000994365 Homo sapiens Integrin alpha-6 Proteins 0.000 claims description 7
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 7
- 101150118944 PRSS1 gene Proteins 0.000 claims description 7
- 102100037265 Podoplanin Human genes 0.000 claims description 7
- 101710118150 Podoplanin Proteins 0.000 claims description 7
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 7
- 102100032491 Serine protease 1 Human genes 0.000 claims description 7
- 239000002875 cyclin dependent kinase inhibitor Substances 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 7
- 210000001116 retinal neuron Anatomy 0.000 claims description 7
- 102100027221 CD81 antigen Human genes 0.000 claims description 6
- 102100037904 CD9 antigen Human genes 0.000 claims description 6
- 102100021260 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Human genes 0.000 claims description 6
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims description 6
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 claims description 6
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 claims description 6
- 101000894906 Homo sapiens Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Proteins 0.000 claims description 6
- 101000606465 Homo sapiens Inactive tyrosine-protein kinase 7 Proteins 0.000 claims description 6
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 claims description 6
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 6
- 102100039813 Inactive tyrosine-protein kinase 7 Human genes 0.000 claims description 6
- 102100032816 Integrin alpha-6 Human genes 0.000 claims description 6
- 102100025304 Integrin beta-1 Human genes 0.000 claims description 6
- 108010009489 Lysosomal-Associated Membrane Protein 3 Proteins 0.000 claims description 6
- 102000002356 Nectin Human genes 0.000 claims description 6
- 108060005251 Nectin Proteins 0.000 claims description 6
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 claims description 6
- 102000001068 Neural Cell Adhesion Molecules Human genes 0.000 claims description 6
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 6
- 239000002771 cell marker Substances 0.000 claims description 6
- 230000006378 damage Effects 0.000 claims description 6
- 108010057417 polysialyl neural cell adhesion molecule Proteins 0.000 claims description 6
- 239000011435 rock Substances 0.000 claims description 6
- 101710137010 Retinol-binding protein 3 Proteins 0.000 claims description 5
- 102100038247 Retinol-binding protein 3 Human genes 0.000 claims description 5
- 102100040756 Rhodopsin Human genes 0.000 claims description 5
- 208000027418 Wounds and injury Diseases 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 claims description 4
- 102100025615 Gamma-synuclein Human genes 0.000 claims description 4
- 101100435109 Homo sapiens PRNP gene Proteins 0.000 claims description 4
- 108090000820 Rhodopsin Proteins 0.000 claims description 4
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 4
- 101000787273 Homo sapiens Gamma-synuclein Proteins 0.000 claims description 3
- 102100024392 Insulin gene enhancer protein ISL-1 Human genes 0.000 claims description 3
- 230000007850 degeneration Effects 0.000 claims description 3
- 108090001121 gamma-Synuclein Proteins 0.000 claims description 3
- 229940126074 CDK kinase inhibitor Drugs 0.000 claims description 2
- 102100034770 Cyclin-dependent kinase inhibitor 3 Human genes 0.000 claims description 2
- 101000945639 Homo sapiens Cyclin-dependent kinase inhibitor 3 Proteins 0.000 claims description 2
- 101001053263 Homo sapiens Insulin gene enhancer protein ISL-1 Proteins 0.000 claims description 2
- 102000010175 Opsin Human genes 0.000 claims description 2
- 108050001704 Opsin Proteins 0.000 claims description 2
- 239000011324 bead Substances 0.000 claims description 2
- 101001051490 Homo sapiens Neural cell adhesion molecule L1 Proteins 0.000 claims 10
- 101000648544 Homo sapiens Sushi domain-containing protein 2 Proteins 0.000 claims 8
- XJBQVBPGIOOPMP-HNNXBMFYSA-N (3s)-3-[[6-[[[3-(methylsulfonylcarbamoyl)phenyl]sulfonylamino]methyl]pyridine-3-carbonyl]amino]-4-oxobutanoic acid Chemical compound CS(=O)(=O)NC(=O)C1=CC=CC(S(=O)(=O)NCC=2N=CC(=CC=2)C(=O)N[C@@H](CC(O)=O)C=O)=C1 XJBQVBPGIOOPMP-HNNXBMFYSA-N 0.000 claims 1
- 101100154912 Mus musculus Tyrp1 gene Proteins 0.000 claims 1
- 210000000130 stem cell Anatomy 0.000 abstract description 20
- 239000003814 drug Substances 0.000 abstract description 9
- 101710175383 Sushi domain-containing protein 2 Proteins 0.000 description 53
- 108090000623 proteins and genes Proteins 0.000 description 43
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 38
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 35
- 239000003550 marker Substances 0.000 description 35
- 102000004169 proteins and genes Human genes 0.000 description 32
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 description 31
- 235000018102 proteins Nutrition 0.000 description 31
- 239000000306 component Substances 0.000 description 30
- 210000000608 photoreceptor cell Anatomy 0.000 description 29
- 108091007433 antigens Proteins 0.000 description 27
- 102000036639 antigens Human genes 0.000 description 27
- 230000037361 pathway Effects 0.000 description 27
- 239000000427 antigen Substances 0.000 description 26
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 25
- 239000006285 cell suspension Substances 0.000 description 24
- 230000008672 reprogramming Effects 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 24
- 239000013598 vector Substances 0.000 description 24
- FHYUGAJXYORMHI-UHFFFAOYSA-N SB 431542 Chemical compound C1=CC(C(=O)N)=CC=C1C1=NC(C=2C=C3OCOC3=CC=2)=C(C=2N=CC=CC=2)N1 FHYUGAJXYORMHI-UHFFFAOYSA-N 0.000 description 23
- 210000001778 pluripotent stem cell Anatomy 0.000 description 22
- 210000001082 somatic cell Anatomy 0.000 description 21
- 208000002780 macular degeneration Diseases 0.000 description 20
- 210000002966 serum Anatomy 0.000 description 20
- 239000012580 N-2 Supplement Substances 0.000 description 19
- 150000001875 compounds Chemical class 0.000 description 19
- 229940054269 sodium pyruvate Drugs 0.000 description 19
- 239000012583 B-27 Supplement Substances 0.000 description 18
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 239000000047 product Substances 0.000 description 18
- 238000000684 flow cytometry Methods 0.000 description 17
- DWJXYEABWRJFSP-XOBRGWDASA-N DAPT Chemical group N([C@@H](C)C(=O)N[C@H](C(=O)OC(C)(C)C)C=1C=CC=CC=1)C(=O)CC1=CC(F)=CC(F)=C1 DWJXYEABWRJFSP-XOBRGWDASA-N 0.000 description 16
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 16
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 16
- 238000011977 dual antiplatelet therapy Methods 0.000 description 16
- 239000010410 layer Substances 0.000 description 16
- 239000011159 matrix material Substances 0.000 description 16
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 16
- 239000000872 buffer Substances 0.000 description 15
- 150000007523 nucleic acids Chemical class 0.000 description 15
- 238000002054 transplantation Methods 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 14
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 14
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 14
- 101150081664 PAX6 gene Proteins 0.000 description 14
- 102000054766 genetic haplotypes Human genes 0.000 description 14
- 210000001525 retina Anatomy 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 13
- 229940121396 wnt pathway inhibitor Drugs 0.000 description 13
- 102000039446 nucleic acids Human genes 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- 108700028369 Alleles Proteins 0.000 description 11
- 230000004186 co-expression Effects 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 101150111214 lin-28 gene Proteins 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 10
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 10
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 10
- 201000007737 Retinal degeneration Diseases 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- 206010064930 age-related macular degeneration Diseases 0.000 description 10
- 201000010099 disease Diseases 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 210000002950 fibroblast Anatomy 0.000 description 10
- 230000002062 proliferating effect Effects 0.000 description 10
- 230000004258 retinal degeneration Effects 0.000 description 10
- 210000003994 retinal ganglion cell Anatomy 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 102100035140 Vitronectin Human genes 0.000 description 9
- 108010031318 Vitronectin Proteins 0.000 description 9
- LZAXPYOBKSJSEX-UHFFFAOYSA-N blebbistatin Chemical compound C1CC2(O)C(=O)C3=CC(C)=CC=C3N=C2N1C1=CC=CC=C1 LZAXPYOBKSJSEX-UHFFFAOYSA-N 0.000 description 9
- 238000012512 characterization method Methods 0.000 description 9
- 238000005138 cryopreservation Methods 0.000 description 9
- 230000018109 developmental process Effects 0.000 description 9
- 238000010494 dissociation reaction Methods 0.000 description 9
- 230000005593 dissociations Effects 0.000 description 9
- 210000001671 embryonic stem cell Anatomy 0.000 description 9
- 210000002744 extracellular matrix Anatomy 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 8
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 8
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 8
- AHJRHEGDXFFMBM-UHFFFAOYSA-N palbociclib Chemical compound N1=C2N(C3CCCC3)C(=O)C(C(=O)C)=C(C)C2=CN=C1NC(N=C1)=CC=C1N1CCNCC1 AHJRHEGDXFFMBM-UHFFFAOYSA-N 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 8
- 102000009027 Albumins Human genes 0.000 description 7
- 108010088751 Albumins Proteins 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 7
- 108010035532 Collagen Proteins 0.000 description 7
- 102000008186 Collagen Human genes 0.000 description 7
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 7
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 7
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 7
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 7
- 102000018697 Membrane Proteins Human genes 0.000 description 7
- 108010052285 Membrane Proteins Proteins 0.000 description 7
- 208000017442 Retinal disease Diseases 0.000 description 7
- 102000040945 Transcription factor Human genes 0.000 description 7
- 108091023040 Transcription factor Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000011575 calcium Substances 0.000 description 7
- 229910052791 calcium Inorganic materials 0.000 description 7
- 230000001413 cellular effect Effects 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 229920001436 collagen Polymers 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 239000003797 essential amino acid Substances 0.000 description 7
- 235000020776 essential amino acid Nutrition 0.000 description 7
- 238000003125 immunofluorescent labeling Methods 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000011777 magnesium Substances 0.000 description 7
- 229910052749 magnesium Inorganic materials 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 241001529453 unidentified herpesvirus Species 0.000 description 7
- 208000010412 Glaucoma Diseases 0.000 description 6
- 101000633984 Homo sapiens Influenza virus NS1A-binding protein Proteins 0.000 description 6
- 101000604411 Homo sapiens NADH-ubiquinone oxidoreductase chain 1 Proteins 0.000 description 6
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 6
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 6
- 210000005156 Müller Glia Anatomy 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 6
- 210000000411 amacrine cell Anatomy 0.000 description 6
- 210000002459 blastocyst Anatomy 0.000 description 6
- 210000002242 embryoid body Anatomy 0.000 description 6
- 239000003102 growth factor Substances 0.000 description 6
- 239000000017 hydrogel Substances 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 210000001161 mammalian embryo Anatomy 0.000 description 6
- 210000005157 neural retina Anatomy 0.000 description 6
- 230000009467 reduction Effects 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 241000701822 Bovine papillomavirus Species 0.000 description 5
- 108010085895 Laminin Proteins 0.000 description 5
- 102000007547 Laminin Human genes 0.000 description 5
- 108050000588 Neurogenic differentiation factor 1 Proteins 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 229920002988 biodegradable polymer Polymers 0.000 description 5
- 239000004621 biodegradable polymer Substances 0.000 description 5
- 230000024245 cell differentiation Effects 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 210000005260 human cell Anatomy 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- JUAVTXYOCISSSL-UHFFFAOYSA-N n-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide;dihydrochloride Chemical compound Cl.Cl.C1=NC=C2C(S(=O)(=O)NCCN)=CC=C(Cl)C2=C1 JUAVTXYOCISSSL-UHFFFAOYSA-N 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229920000728 polyester Polymers 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 230000011664 signaling Effects 0.000 description 5
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 description 4
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 4
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 4
- 108010043324 Amyloid Precursor Protein Secretases Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 101150099612 Esrrb gene Proteins 0.000 description 4
- 108010067306 Fibronectins Proteins 0.000 description 4
- 102000016359 Fibronectins Human genes 0.000 description 4
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 4
- 108010075704 HLA-A Antigens Proteins 0.000 description 4
- 101000773083 Homo sapiens 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 4
- 102000004877 Insulin Human genes 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 108010088225 Nestin Proteins 0.000 description 4
- 102000008730 Nestin Human genes 0.000 description 4
- 229920000954 Polyglycolide Polymers 0.000 description 4
- 229920000331 Polyhydroxybutyrate Polymers 0.000 description 4
- 229940072056 alginate Drugs 0.000 description 4
- 229920000615 alginic acid Polymers 0.000 description 4
- 235000010443 alginic acid Nutrition 0.000 description 4
- KXDAEFPNCMNJSK-UHFFFAOYSA-N benzene carboxamide Natural products NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 4
- 229920000229 biodegradable polyester Polymers 0.000 description 4
- 239000004622 biodegradable polyester Substances 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 210000005055 nestin Anatomy 0.000 description 4
- 238000010899 nucleation Methods 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 239000005015 poly(hydroxybutyrate) Substances 0.000 description 4
- 229920001610 polycaprolactone Polymers 0.000 description 4
- 239000004632 polycaprolactone Substances 0.000 description 4
- 239000004633 polyglycolic acid Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- SYTBZMRGLBWNTM-SNVBAGLBSA-N (R)-flurbiprofen Chemical compound FC1=CC([C@H](C(O)=O)C)=CC=C1C1=CC=CC=C1 SYTBZMRGLBWNTM-SNVBAGLBSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 201000004569 Blindness Diseases 0.000 description 3
- 108091007914 CDKs Proteins 0.000 description 3
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 3
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 3
- 108090000266 Cyclin-dependent kinases Proteins 0.000 description 3
- 102000003903 Cyclin-dependent kinases Human genes 0.000 description 3
- 206010012689 Diabetic retinopathy Diseases 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 208000003098 Ganglion Cysts Diseases 0.000 description 3
- 102100028976 HLA class I histocompatibility antigen, B alpha chain Human genes 0.000 description 3
- 108010058607 HLA-B Antigens Proteins 0.000 description 3
- 108091006905 Human Serum Albumin Proteins 0.000 description 3
- 101000767631 Human papillomavirus type 16 Protein E7 Proteins 0.000 description 3
- 208000032578 Inherited retinal disease Diseases 0.000 description 3
- 201000003533 Leber congenital amaurosis Diseases 0.000 description 3
- 102000043129 MHC class I family Human genes 0.000 description 3
- 108091054437 MHC class I family Proteins 0.000 description 3
- 241000282560 Macaca mulatta Species 0.000 description 3
- 206010061323 Optic neuropathy Diseases 0.000 description 3
- 238000010240 RT-PCR analysis Methods 0.000 description 3
- 206010038848 Retinal detachment Diseases 0.000 description 3
- 208000032430 Retinal dystrophy Diseases 0.000 description 3
- 206010038923 Retinopathy Diseases 0.000 description 3
- 101100465275 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP24 gene Proteins 0.000 description 3
- 208000005400 Synovial Cyst Diseases 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 102000003425 Tyrosinase Human genes 0.000 description 3
- 108060008724 Tyrosinase Proteins 0.000 description 3
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000003915 cell function Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 210000003850 cellular structure Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 238000005206 flow analysis Methods 0.000 description 3
- 201000006321 fundus dystrophy Diseases 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 210000002287 horizontal cell Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 208000017532 inherited retinal dystrophy Diseases 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 239000012533 medium component Substances 0.000 description 3
- 239000011325 microbead Substances 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 210000000287 oocyte Anatomy 0.000 description 3
- 208000020911 optic nerve disease Diseases 0.000 description 3
- 239000000825 pharmaceutical preparation Substances 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 238000007747 plating Methods 0.000 description 3
- 239000004631 polybutylene succinate Substances 0.000 description 3
- 229920002961 polybutylene succinate Polymers 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000004264 retinal detachment Effects 0.000 description 3
- 210000003583 retinal pigment epithelium Anatomy 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000007423 screening assay Methods 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000010374 somatic cell nuclear transfer Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- LXEYYZYDWLAIPW-KBVFCZPLSA-N (2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino]-N-[1-[1-(2,2-dimethylpropylamino)-2-methylpropan-2-yl]imidazol-4-yl]pentanamide dihydrobromide Chemical compound Br.Br.CCC[C@H](N[C@H]1CCc2cc(F)cc(F)c2C1)C(=O)Nc1cn(cn1)C(C)(C)CNCC(C)(C)C LXEYYZYDWLAIPW-KBVFCZPLSA-N 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- QSHGISMANBKLQL-OWJWWREXSA-N (2s)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-n-[(7s)-5-methyl-6-oxo-7h-benzo[d][1]benzazepin-7-yl]propanamide Chemical compound N([C@@H](C)C(=O)N[C@@H]1C(N(C)C2=CC=CC=C2C2=CC=CC=C21)=O)C(=O)CC1=CC(F)=CC(F)=C1 QSHGISMANBKLQL-OWJWWREXSA-N 0.000 description 2
- LAQPKDLYOBZWBT-NYLDSJSYSA-N (2s,4s,5r,6r)-5-acetamido-2-{[(2s,3r,4s,5s,6r)-2-{[(2r,3r,4r,5r)-5-acetamido-1,2-dihydroxy-6-oxo-4-{[(2s,3s,4r,5s,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}hexan-3-yl]oxy}-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy}-4-hydroxy-6-[(1r,2r)-1,2,3-trihydrox Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 LAQPKDLYOBZWBT-NYLDSJSYSA-N 0.000 description 2
- IZAOBRWCUGOKNH-OAHLLOKOSA-N 4-[2-[(1R)-1-(N-(4-chlorophenyl)sulfonyl-2,5-difluoroanilino)ethyl]-5-fluorophenyl]butanoic acid Chemical compound C=1C=C(Cl)C=CC=1S(=O)(=O)N([C@H](C)C=1C(=CC(F)=CC=1)CCCC(O)=O)C1=CC(F)=CC=C1F IZAOBRWCUGOKNH-OAHLLOKOSA-N 0.000 description 2
- PSXOKXJMVRSARX-SCSAIBSYSA-N 5-chloro-n-[(2s)-4,4,4-trifluoro-1-hydroxy-3-(trifluoromethyl)butan-2-yl]thiophene-2-sulfonamide Chemical compound FC(F)(F)C(C(F)(F)F)[C@@H](CO)NS(=O)(=O)C1=CC=C(Cl)S1 PSXOKXJMVRSARX-SCSAIBSYSA-N 0.000 description 2
- 102000018918 Activin Receptors Human genes 0.000 description 2
- 108010052946 Activin Receptors Proteins 0.000 description 2
- 102100026440 Arrestin-C Human genes 0.000 description 2
- 208000037663 Best vitelliform macular dystrophy Diseases 0.000 description 2
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 2
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 2
- 229940124297 CDK 4/6 inhibitor Drugs 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 2
- 206010010356 Congenital anomaly Diseases 0.000 description 2
- 229920001634 Copolyester Polymers 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101150033269 ESRRG gene Proteins 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 102100023371 Forkhead box protein N1 Human genes 0.000 description 2
- 102000002254 Glycogen Synthase Kinase 3 Human genes 0.000 description 2
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000907576 Homo sapiens Forkhead box protein N1 Proteins 0.000 description 2
- 101000988591 Homo sapiens Minor histocompatibility antigen H13 Proteins 0.000 description 2
- 101000912686 Homo sapiens Probable ATP-dependent RNA helicase DDX23 Proteins 0.000 description 2
- 101000976622 Homo sapiens Zinc finger protein 42 homolog Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 101100540311 Human papillomavirus type 16 E6 gene Proteins 0.000 description 2
- 108700005092 MHC Class II Genes Proteins 0.000 description 2
- 102000043131 MHC class II family Human genes 0.000 description 2
- 108091054438 MHC class II family Proteins 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 208000035719 Maculopathy Diseases 0.000 description 2
- 102100037812 Medium-wave-sensitive opsin 1 Human genes 0.000 description 2
- 102100029083 Minor histocompatibility antigen H13 Human genes 0.000 description 2
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 2
- 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- WRKPZSMRWPJJDH-UHFFFAOYSA-N N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thio]acetamide Chemical compound S1C2=CC(C)=CC=C2N=C1NC(=O)CSC1=NC=2CCSC=2C(=O)N1C1=CC=CC=C1 WRKPZSMRWPJJDH-UHFFFAOYSA-N 0.000 description 2
- HEKAIDKUDLCBRU-UHFFFAOYSA-N N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide Chemical compound S1C(CC)=NC(C=2C=C(C)C=CC=2)=C1C(C=1)=CC=NC=1NC(=O)C1=CC=CC=C1 HEKAIDKUDLCBRU-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 206010036590 Premature baby Diseases 0.000 description 2
- 102100026136 Probable ATP-dependent RNA helicase DDX23 Human genes 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- 208000004453 Retinal Dysplasia Diseases 0.000 description 2
- 206010057430 Retinal injury Diseases 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000701062 Saimiriine gammaherpesvirus 2 Species 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 208000027073 Stargardt disease Diseases 0.000 description 2
- KLGQSVMIPOVQAX-UHFFFAOYSA-N XAV939 Chemical compound N=1C=2CCSCC=2C(O)=NC=1C1=CC=C(C(F)(F)F)C=C1 KLGQSVMIPOVQAX-UHFFFAOYSA-N 0.000 description 2
- 102100023550 Zinc finger protein 42 homolog Human genes 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 229950008971 begacestat Drugs 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229920001400 block copolymer Polymers 0.000 description 2
- 229940112869 bone morphogenetic protein Drugs 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000003483 chromatin Anatomy 0.000 description 2
- 201000006754 cone-rod dystrophy Diseases 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 208000030533 eye disease Diseases 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 239000010408 film Substances 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000003540 gamma secretase inhibitor Substances 0.000 description 2
- 102000004963 gamma-Synuclein Human genes 0.000 description 2
- 238000001476 gene delivery Methods 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 108010028309 kalinin Proteins 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920005615 natural polymer Polymers 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 230000004031 neuronal differentiation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 210000001743 on-bipolar cell Anatomy 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 229920000747 poly(lactic acid) Polymers 0.000 description 2
- 229920002627 poly(phosphazenes) Polymers 0.000 description 2
- 229920000058 polyacrylate Polymers 0.000 description 2
- 229920006149 polyester-amide block copolymer Polymers 0.000 description 2
- 239000004626 polylactic acid Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011165 process development Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011552 rat model Methods 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000004283 retinal dysfunction Effects 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- CYOHGALHFOKKQC-UHFFFAOYSA-N selumetinib Chemical compound OCCONC(=O)C=1C=C2N(C)C=NC2=C(F)C=1NC1=CC=C(Br)C=C1Cl CYOHGALHFOKKQC-UHFFFAOYSA-N 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000030968 tissue homeostasis Effects 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 230000002110 toxicologic effect Effects 0.000 description 2
- 231100000723 toxicological property Toxicity 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- 201000007790 vitelliform macular dystrophy Diseases 0.000 description 2
- 208000020938 vitelliform macular dystrophy 2 Diseases 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- BPNUQXPIQBZCMR-IBGZPJMESA-N (2s)-1-{[5-(3-methyl-1h-indazol-5-yl)pyridin-3-yl]oxy}-3-phenylpropan-2-amine Chemical compound C([C@H](N)COC=1C=NC=C(C=1)C1=CC=C2NN=C(C2=C1)C)C1=CC=CC=C1 BPNUQXPIQBZCMR-IBGZPJMESA-N 0.000 description 1
- XQQUSYWGKLRJRA-RABCQHRBSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s,3s)-2-amino-3-methylpentanoyl]amino]hexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-3-methylbutanoic acid Chemical group CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XQQUSYWGKLRJRA-RABCQHRBSA-N 0.000 description 1
- SCFMWQIQBVZOQR-UHFFFAOYSA-N (4-butoxy-1h-pyrazolo[3,4-b]pyridin-5-yl)-(2,6-difluoro-4-methylphenyl)methanone Chemical compound C1=NC=2NN=CC=2C(OCCCC)=C1C(=O)C1=C(F)C=C(C)C=C1F SCFMWQIQBVZOQR-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- LBPKYPYHDKKRFS-UHFFFAOYSA-N 1,5-naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1h-pyrazol-4-yl]- Chemical compound CC1=CC=CC(C2=C(C=NN2)C=2N=C3C=CC=NC3=CC=2)=N1 LBPKYPYHDKKRFS-UHFFFAOYSA-N 0.000 description 1
- YTOPFCCWCSOHFV-UHFFFAOYSA-N 2,6-dimethyl-4-tridecylmorpholine Chemical compound CCCCCCCCCCCCCN1CC(C)OC(C)C1 YTOPFCCWCSOHFV-UHFFFAOYSA-N 0.000 description 1
- PIMQWRZWLQKKBJ-SFHVURJKSA-N 2-[(2S)-1-[3-ethyl-7-[(1-oxido-3-pyridin-1-iumyl)methylamino]-5-pyrazolo[1,5-a]pyrimidinyl]-2-piperidinyl]ethanol Chemical compound C=1C(N2[C@@H](CCCC2)CCO)=NC2=C(CC)C=NN2C=1NCC1=CC=C[N+]([O-])=C1 PIMQWRZWLQKKBJ-SFHVURJKSA-N 0.000 description 1
- DKXHSOUZPMHNIZ-UHFFFAOYSA-N 2-pyridin-4-yl-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one Chemical compound C=1C=2C(=O)NCCC=2NC=1C1=CC=NC=C1 DKXHSOUZPMHNIZ-UHFFFAOYSA-N 0.000 description 1
- 238000012605 2D cell culture Methods 0.000 description 1
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 1
- JOSXKPZXMVHRKU-UHFFFAOYSA-N 3,5-bis(4-nitrophenoxy)benzoic acid Chemical compound C=1C(OC=2C=CC(=CC=2)[N+]([O-])=O)=CC(C(=O)O)=CC=1OC1=CC=C([N+]([O-])=O)C=C1 JOSXKPZXMVHRKU-UHFFFAOYSA-N 0.000 description 1
- 125000004211 3,5-difluorophenyl group Chemical group [H]C1=C(F)C([H])=C(*)C([H])=C1F 0.000 description 1
- WJRRGYBTGDJBFX-UHFFFAOYSA-N 4-(2-methyl-3-propan-2-yl-4-imidazolyl)-N-(4-methylsulfonylphenyl)-2-pyrimidinamine Chemical compound CC(C)N1C(C)=NC=C1C1=CC=NC(NC=2C=CC(=CC=2)S(C)(=O)=O)=N1 WJRRGYBTGDJBFX-UHFFFAOYSA-N 0.000 description 1
- DXLXRNZCYAYUED-UHFFFAOYSA-N 4-[2-[4-(3-quinolin-4-ylpyrazolo[1,5-a]pyrimidin-6-yl)phenoxy]ethyl]morpholine Chemical compound C=1C=C(C2=CN3N=CC(=C3N=C2)C=2C3=CC=CC=C3N=CC=2)C=CC=1OCCN1CCOCC1 DXLXRNZCYAYUED-UHFFFAOYSA-N 0.000 description 1
- KDKUVYLMPJIGKA-UHFFFAOYSA-N 4-[[5-amino-1-[(2,6-difluorophenyl)-oxomethyl]-1,2,4-triazol-3-yl]amino]benzenesulfonamide Chemical compound N=1N(C(=O)C=2C(=CC=CC=2F)F)C(N)=NC=1NC1=CC=C(S(N)(=O)=O)C=C1 KDKUVYLMPJIGKA-UHFFFAOYSA-N 0.000 description 1
- AMDGKLWVCUXONP-UHFFFAOYSA-N 7-amino-4-chloro-3-methoxy-2-benzopyran-1-one Chemical compound NC1=CC=C2C(Cl)=C(OC)OC(=O)C2=C1 AMDGKLWVCUXONP-UHFFFAOYSA-N 0.000 description 1
- 108010059616 Activins Proteins 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 1
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 1
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 1
- 101000654470 Arabidopsis thaliana Signal peptide peptidase Proteins 0.000 description 1
- 108050003620 Arrestin-C Proteins 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- KHBQMWCZKVMBLN-UHFFFAOYSA-N Benzenesulfonamide Chemical compound NS(=O)(=O)C1=CC=CC=C1 KHBQMWCZKVMBLN-UHFFFAOYSA-N 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 102100025142 Beta-microseminoprotein Human genes 0.000 description 1
- 102000004152 Bone morphogenetic protein 1 Human genes 0.000 description 1
- 108090000654 Bone morphogenetic protein 1 Proteins 0.000 description 1
- 102100028726 Bone morphogenetic protein 10 Human genes 0.000 description 1
- 102100028727 Bone morphogenetic protein 15 Human genes 0.000 description 1
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 1
- 102100024504 Bone morphogenetic protein 3 Human genes 0.000 description 1
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 1
- 102100022526 Bone morphogenetic protein 5 Human genes 0.000 description 1
- 102100022525 Bone morphogenetic protein 6 Human genes 0.000 description 1
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 1
- 101100161935 Caenorhabditis elegans act-4 gene Proteins 0.000 description 1
- 101100257372 Caenorhabditis elegans sox-3 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- UDHXJZHVNHGCEC-UHFFFAOYSA-N Chlorophacinone Chemical compound C1=CC(Cl)=CC=C1C(C=1C=CC=CC=1)C(=O)C1C(=O)C2=CC=CC=C2C1=O UDHXJZHVNHGCEC-UHFFFAOYSA-N 0.000 description 1
- 208000030275 Chondronectin Diseases 0.000 description 1
- 208000033810 Choroidal dystrophy Diseases 0.000 description 1
- ODRITQGYYWHQGM-INIZCTEOSA-N ClC1=CC=C(C=C1)[C@H]1N(CCC1)C(=O)C=1C=C2C(=NC=1)NN=C2C Chemical compound ClC1=CC=C(C=C1)[C@H]1N(CCC1)C(=O)C=1C=C2C(=NC=1)NN=C2C ODRITQGYYWHQGM-INIZCTEOSA-N 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102100024203 Collagen alpha-1(XIV) chain Human genes 0.000 description 1
- 101710106877 Collagen alpha-1(XIV) chain Proteins 0.000 description 1
- 208000006992 Color Vision Defects Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010003730 Cone Opsins Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 108010024986 Cyclin-Dependent Kinase 2 Proteins 0.000 description 1
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 1
- 108010025454 Cyclin-Dependent Kinase 5 Proteins 0.000 description 1
- 108010025468 Cyclin-Dependent Kinase 6 Proteins 0.000 description 1
- 102100032857 Cyclin-dependent kinase 1 Human genes 0.000 description 1
- 101710106279 Cyclin-dependent kinase 1 Proteins 0.000 description 1
- 102100036239 Cyclin-dependent kinase 2 Human genes 0.000 description 1
- 102100036329 Cyclin-dependent kinase 3 Human genes 0.000 description 1
- 102100036252 Cyclin-dependent kinase 4 Human genes 0.000 description 1
- 102100026804 Cyclin-dependent kinase 6 Human genes 0.000 description 1
- 102100026810 Cyclin-dependent kinase 7 Human genes 0.000 description 1
- 102100024456 Cyclin-dependent kinase 8 Human genes 0.000 description 1
- 102100024457 Cyclin-dependent kinase 9 Human genes 0.000 description 1
- 102100026805 Cyclin-dependent-like kinase 5 Human genes 0.000 description 1
- JMIFGARJSWXZSH-UHFFFAOYSA-N DMH1 Chemical compound C1=CC(OC(C)C)=CC=C1C1=CN2N=CC(C=3C4=CC=CC=C4N=CC=3)=C2N=C1 JMIFGARJSWXZSH-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102100031480 Dual specificity mitogen-activated protein kinase kinase 1 Human genes 0.000 description 1
- 101710146526 Dual specificity mitogen-activated protein kinase kinase 1 Proteins 0.000 description 1
- 102100023266 Dual specificity mitogen-activated protein kinase kinase 2 Human genes 0.000 description 1
- 101710146529 Dual specificity mitogen-activated protein kinase kinase 2 Proteins 0.000 description 1
- 108010031111 EBV-encoded nuclear antigen 1 Proteins 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 102000004437 G-Protein-Coupled Receptor Kinase 1 Human genes 0.000 description 1
- 108091004242 G-Protein-Coupled Receptor Kinase 1 Proteins 0.000 description 1
- 241000701047 Gallid alphaherpesvirus 2 Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 101710201754 Green-sensitive opsin Proteins 0.000 description 1
- 208000007698 Gyrate Atrophy Diseases 0.000 description 1
- 102100028971 HLA class I histocompatibility antigen, C alpha chain Human genes 0.000 description 1
- 108010052199 HLA-C Antigens Proteins 0.000 description 1
- 108010010378 HLA-DP Antigens Proteins 0.000 description 1
- 102000015789 HLA-DP Antigens Human genes 0.000 description 1
- 108010062347 HLA-DQ Antigens Proteins 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 101000785755 Homo sapiens Arrestin-C Proteins 0.000 description 1
- 101000576812 Homo sapiens Beta-microseminoprotein Proteins 0.000 description 1
- 101000695367 Homo sapiens Bone morphogenetic protein 10 Proteins 0.000 description 1
- 101000695360 Homo sapiens Bone morphogenetic protein 15 Proteins 0.000 description 1
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 description 1
- 101000762375 Homo sapiens Bone morphogenetic protein 3 Proteins 0.000 description 1
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 description 1
- 101000899388 Homo sapiens Bone morphogenetic protein 5 Proteins 0.000 description 1
- 101000899390 Homo sapiens Bone morphogenetic protein 6 Proteins 0.000 description 1
- 101000899361 Homo sapiens Bone morphogenetic protein 7 Proteins 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 101000715946 Homo sapiens Cyclin-dependent kinase 3 Proteins 0.000 description 1
- 101000911952 Homo sapiens Cyclin-dependent kinase 7 Proteins 0.000 description 1
- 101000980937 Homo sapiens Cyclin-dependent kinase 8 Proteins 0.000 description 1
- 101000980930 Homo sapiens Cyclin-dependent kinase 9 Proteins 0.000 description 1
- 101000598987 Homo sapiens Medium-wave-sensitive opsin 1 Proteins 0.000 description 1
- 101000580370 Homo sapiens RAD52 motif-containing protein 1 Proteins 0.000 description 1
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 241000701806 Human papillomavirus Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 102100026818 Inhibin beta E chain Human genes 0.000 description 1
- 108010004250 Inhibins Proteins 0.000 description 1
- 102000002746 Inhibins Human genes 0.000 description 1
- 108010028750 Integrin-Binding Sialoprotein Proteins 0.000 description 1
- 102000016921 Integrin-Binding Sialoprotein Human genes 0.000 description 1
- 108091006975 Iron transporters Proteins 0.000 description 1
- 101150070110 Isl1 gene Proteins 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 101150072501 Klf2 gene Proteins 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 102100022744 Laminin subunit alpha-3 Human genes 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 102000002092 Left-Right Determination Factor Human genes 0.000 description 1
- 108050009437 Left-Right Determination Factor Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 230000037364 MAPK/ERK pathway Effects 0.000 description 1
- 208000001344 Macular Edema Diseases 0.000 description 1
- 206010025415 Macular oedema Diseases 0.000 description 1
- 101710123538 Medium-wave-sensitive opsin 1 Proteins 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100310648 Mus musculus Sox17 gene Proteins 0.000 description 1
- 101100310650 Mus musculus Sox18 gene Proteins 0.000 description 1
- 101100257376 Mus musculus Sox3 gene Proteins 0.000 description 1
- 101000976618 Mus musculus Zinc finger protein 42 Proteins 0.000 description 1
- 208000024080 Myopic macular degeneration Diseases 0.000 description 1
- OUSFTKFNBAZUKL-UHFFFAOYSA-N N-(5-{[(5-tert-butyl-1,3-oxazol-2-yl)methyl]sulfanyl}-1,3-thiazol-2-yl)piperidine-4-carboxamide Chemical compound O1C(C(C)(C)C)=CN=C1CSC(S1)=CN=C1NC(=O)C1CCNCC1 OUSFTKFNBAZUKL-UHFFFAOYSA-N 0.000 description 1
- HUXYBQXJVXOMKX-UHFFFAOYSA-N N-[6,6-dimethyl-5-[(1-methyl-4-piperidinyl)-oxomethyl]-1,4-dihydropyrrolo[3,4-c]pyrazol-3-yl]-3-methylbutanamide Chemical compound CC(C)CC(=O)NC1=NNC(C2(C)C)=C1CN2C(=O)C1CCN(C)CC1 HUXYBQXJVXOMKX-UHFFFAOYSA-N 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 241001045988 Neogene Species 0.000 description 1
- 102100037369 Nidogen-1 Human genes 0.000 description 1
- 208000001140 Night Blindness Diseases 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 208000022873 Ocular disease Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 208000030768 Optic nerve injury Diseases 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 108010081689 Osteopontin Proteins 0.000 description 1
- 102000004264 Osteopontin Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 208000034247 Pattern dystrophy Diseases 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 208000005107 Premature Birth Diseases 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 1
- 102100027420 RAD52 motif-containing protein 1 Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 208000002367 Retinal Perforations Diseases 0.000 description 1
- 206010038910 Retinitis Diseases 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 108090000799 Rhodopsin kinases Proteins 0.000 description 1
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 1
- 241000235343 Saccharomycetales Species 0.000 description 1
- 102100030053 Secreted frizzled-related protein 3 Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 101150001847 Sox15 gene Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000272534 Struthio camelus Species 0.000 description 1
- 108010008125 Tenascin Proteins 0.000 description 1
- 102000007000 Tenascin Human genes 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 201000005485 Toxoplasmosis Diseases 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 108091005906 Type I transmembrane proteins Proteins 0.000 description 1
- 206010053648 Vascular occlusion Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 108010020277 WD repeat containing planar cell polarity effector Proteins 0.000 description 1
- 101150010310 WNT-4 gene Proteins 0.000 description 1
- 101150019524 WNT2 gene Proteins 0.000 description 1
- 102000052556 Wnt-2 Human genes 0.000 description 1
- 108700020986 Wnt-2 Proteins 0.000 description 1
- 102000052549 Wnt-3 Human genes 0.000 description 1
- 108700020985 Wnt-3 Proteins 0.000 description 1
- 102000052548 Wnt-4 Human genes 0.000 description 1
- 108700020984 Wnt-4 Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- WNLRTRBMVRJNCN-UHFFFAOYSA-L adipate(2-) Chemical compound [O-]C(=O)CCCCC([O-])=O WNLRTRBMVRJNCN-UHFFFAOYSA-L 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- BIIVYFLTOXDAOV-YVEFUNNKSA-N alvocidib Chemical compound O[C@@H]1CN(C)CC[C@@H]1C1=C(O)C=C(O)C2=C1OC(C=1C(=CC=CC=1)Cl)=CC2=O BIIVYFLTOXDAOV-YVEFUNNKSA-N 0.000 description 1
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical group C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- METKIMKYRPQLGS-UHFFFAOYSA-N atenolol Chemical compound CC(C)NCC(O)COC1=CC=C(CC(N)=O)C=C1 METKIMKYRPQLGS-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- ACWZRVQXLIRSDF-UHFFFAOYSA-N binimetinib Chemical compound OCCONC(=O)C=1C=C2N(C)C=NC2=C(F)C=1NC1=CC=C(Br)C=C1F ACWZRVQXLIRSDF-UHFFFAOYSA-N 0.000 description 1
- 239000000227 bioadhesive Substances 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000004703 blastocyst inner cell mass Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- DQXBYHZEEUGOBF-UHFFFAOYSA-N but-3-enoic acid;ethene Chemical compound C=C.OC(=O)CC=C DQXBYHZEEUGOBF-UHFFFAOYSA-N 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000003986 cell retinal photoreceptor Anatomy 0.000 description 1
- 230000017455 cell-cell adhesion Effects 0.000 description 1
- 230000035289 cell-matrix adhesion Effects 0.000 description 1
- 210000003570 cell-matrix junction Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000003161 choroid Anatomy 0.000 description 1
- 208000003571 choroideremia Diseases 0.000 description 1
- 239000011247 coating layer Substances 0.000 description 1
- 239000000512 collagen gel Substances 0.000 description 1
- 239000000515 collagen sponge Substances 0.000 description 1
- 201000007254 color blindness Diseases 0.000 description 1
- 230000004456 color vision Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 201000008615 cone dystrophy Diseases 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000002826 coolant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- XHBVYDAKJHETMP-UHFFFAOYSA-N dorsomorphin Chemical compound C=1C=C(C2=CN3N=CC(=C3N=C2)C=2C=CN=CC=2)C=CC=1OCCN1CCCCC1 XHBVYDAKJHETMP-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 108010015749 epinectin Proteins 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 108060002895 fibrillin Proteins 0.000 description 1
- 102000013370 fibrillin Human genes 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000036433 growing body Effects 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000012615 high-resolution technique Methods 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 108700020610 human chondronectin Proteins 0.000 description 1
- 102000043667 human chondronectin Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- DKAGJZJALZXOOV-UHFFFAOYSA-N hydrate;hydrochloride Chemical compound O.Cl DKAGJZJALZXOOV-UHFFFAOYSA-N 0.000 description 1
- 239000000416 hydrocolloid Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 150000001261 hydroxy acids Chemical class 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010949 in-process test method Methods 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010088381 isoleucyl-lysyl-valyl-alanyl-valine Chemical group 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 108700041430 link Proteins 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 208000018769 loss of vision Diseases 0.000 description 1
- 231100000864 loss of vision Toxicity 0.000 description 1
- 208000029233 macular holes Diseases 0.000 description 1
- 201000010230 macular retinal edema Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 239000002991 molded plastic Substances 0.000 description 1
- 230000001002 morphogenetic effect Effects 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- RXZMYLDMFYNEIM-UHFFFAOYSA-N n,1,4,4-tetramethyl-8-[4-(4-methylpiperazin-1-yl)anilino]-5h-pyrazolo[4,3-h]quinazoline-3-carboxamide Chemical compound CNC(=O)C1=NN(C)C(C2=N3)=C1C(C)(C)CC2=CN=C3NC(C=C1)=CC=C1N1CCN(C)CC1 RXZMYLDMFYNEIM-UHFFFAOYSA-N 0.000 description 1
- JJEDWBQZCRESJL-DEDYPNTBSA-N n-[(e)-(5-methylfuran-2-yl)methylideneamino]-2-phenoxybenzamide Chemical compound O1C(C)=CC=C1\C=N\NC(=O)C1=CC=CC=C1OC1=CC=CC=C1 JJEDWBQZCRESJL-DEDYPNTBSA-N 0.000 description 1
- RDSACQWTXKSHJT-NSHDSACASA-N n-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2s)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide Chemical compound C1CC1(C[C@H](O)CO)S(=O)(=O)NC=1C(OC)=CC(F)=C(F)C=1NC1=CC=C(I)C=C1F RDSACQWTXKSHJT-NSHDSACASA-N 0.000 description 1
- UZWDCWONPYILKI-UHFFFAOYSA-N n-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine Chemical compound C1CN(CC)CCN1CC(C=N1)=CC=C1NC1=NC=C(F)C(C=2C=C3N(C(C)C)C(C)=NC3=C(F)C=2)=N1 UZWDCWONPYILKI-UHFFFAOYSA-N 0.000 description 1
- 108700043045 nanoluc Proteins 0.000 description 1
- 101150091879 neo gene Proteins 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 108010008217 nidogen Proteins 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 208000014380 ornithine aminotransferase deficiency Diseases 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229940118537 p53 inhibitor Drugs 0.000 description 1
- 229960004390 palbociclib Drugs 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- IPWFJLQDVFKJDU-UHFFFAOYSA-N pentanamide Chemical compound CCCCC(N)=O IPWFJLQDVFKJDU-UHFFFAOYSA-N 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000016732 phototransduction Effects 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000903 polyhydroxyalkanoate Polymers 0.000 description 1
- 229920000193 polymethacrylate Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- XELRMPRLCPFTBH-UHFFFAOYSA-N quinazoline-2,4-diamine Chemical compound C1=CC=CC2=NC(N)=NC(N)=C21 XELRMPRLCPFTBH-UHFFFAOYSA-N 0.000 description 1
- 108091006082 receptor inhibitors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000000964 retinal cone photoreceptor cell Anatomy 0.000 description 1
- 230000004243 retinal function Effects 0.000 description 1
- 210000003432 retinal horizontal cell Anatomy 0.000 description 1
- 239000000790 retinal pigment Substances 0.000 description 1
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 1
- 210000000880 retinal rod photoreceptor cell Anatomy 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000003590 rho kinase inhibitor Substances 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000009118 salvage therapy Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- BTIHMVBBUGXLCJ-OAHLLOKOSA-N seliciclib Chemical compound C=12N=CN(C(C)C)C2=NC(N[C@@H](CO)CC)=NC=1NCC1=CC=CC=C1 BTIHMVBBUGXLCJ-OAHLLOKOSA-N 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 102000035025 signaling receptors Human genes 0.000 description 1
- 108091005475 signaling receptors Proteins 0.000 description 1
- 102000035087 single-pass transmembrane proteins Human genes 0.000 description 1
- 108091005496 single-pass transmembrane proteins Proteins 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 210000001988 somatic stem cell Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000004876 tela submucosa Anatomy 0.000 description 1
- SJMDMGHPMLKLHQ-UHFFFAOYSA-N tert-butyl 2-aminoacetate Chemical compound CC(C)(C)OC(=O)CN SJMDMGHPMLKLHQ-UHFFFAOYSA-N 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 229940035024 thioglycerol Drugs 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 229960004066 trametinib Drugs 0.000 description 1
- LIRYPHYGHXZJBZ-UHFFFAOYSA-N trametinib Chemical compound CC(=O)NC1=CC=CC(N2C(N(C3CC3)C(=O)C3=C(NC=4C(=CC(I)=CC=4)F)N(C)C(=O)C(C)=C32)=O)=C1 LIRYPHYGHXZJBZ-UHFFFAOYSA-N 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 210000000143 trophectoderm cell Anatomy 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000002477 vacuolizing effect Effects 0.000 description 1
- 239000012808 vapor phase Substances 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 208000021331 vascular occlusion disease Diseases 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/105—Insulin-like growth factors [IGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/73—Hydrolases (EC 3.)
- C12N2501/734—Proteases (EC 3.4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Abstract
Provided herein are methods of producing a photoreceptor precursor (PRP) cell population derived from stem cells. Further provided herein are methods of using the PRP cell populations, such as for therapeutics.
Description
DESCRIPTION
METHOD FOR DIFFERENTIATION OF OCULAR CELLS AND USE THEREOF
[0001] This application claims the benefit of United States Provisional Patent Application No. 62/660,899, filed April 20, 2018, which is incorporated herein by reference in their entirety.
BACKGROUND
1. Field
METHOD FOR DIFFERENTIATION OF OCULAR CELLS AND USE THEREOF
[0001] This application claims the benefit of United States Provisional Patent Application No. 62/660,899, filed April 20, 2018, which is incorporated herein by reference in their entirety.
BACKGROUND
1. Field
[0002] The present disclosure relates generally to the field of stem cell biology. More particularly, it concerns methods of differentiating pluripotent stem cells to various ocular cells, including photoreceptor precursor (PRP) cells.
2. Description of Related Art
2. Description of Related Art
[0003] The retina is a light-sensitive layer of tissue that lines the inner surface of the eye. Photoreceptor cells, either rods or cones, in the retina are directly sensitive to light and transform chemical light signals into electrical events that trigger nerve impulses. The impaired or complete loss of function of photoreceptor cells is one of the causes of irreversible blindness in retinal diseases, such as inherited retinal degenerations and age-related macular degeneration (AMD). Retinal ganglion cell (RGC) death in glaucoma also results in irreversible loss of vision. Rescuing the degenerated retina is a major challenge and cell replacement is one of the most promising approaches (Pearson etal., 2012).
[0004] The production of induced pluripotent stem cells (iPSCs) from adult somatic mouse cells in 2006 provided an important breakthrough for stem cell research, drug development, models of disease, and cellular therapeutics (Takahashi et al., 2006). Human iPSCs can be differentiated to specialized cell types and have the potential for patient-specific, immune-matched cells for regenerative medicine (Yu et al., 2007). The use of human pluripotent stem cells, embryonic stem (ES) cells and iPSCs opens up a new avenue for treating human retinal degenerative diseases.
[0005] Human ES (hES) and iPS (hiPS) cells that have the ability to be expanded indefinitely in culture while retaining their pluripotent status could be used as an unlimited source of photoreceptor cells for tissue transplantation (Comyn etal., 2010).
A growing body of convergent data demonstrated hES cell neural retina commitment potential after embryoid body formation, and capacity to further differentiate into cells expressing photoreceptor markers (Zhong etal., 2014; Meyer etal., 2009). The different methods previously developed, though real advances, still suffer from drawbacks generally associated with pluripotent stem cell differentiation into highly specialized cell types. These protocols for photoreceptor-directed differentiation of hES or hiPS cells require several steps, addition of several molecules, and are rather inefficient. Current methods for differentiation into the photoreceptor cell lineage require the formation of embryoid bodies and/or require manual selection of retinal cells from culture. Thus, there is a need in the art for efficient and reliable large-scale methods for obtaining substantially pure cultures of certain human neuroepithelial lineage cells, particularly photoreceptor precursor (PRP) cells.
SUMMARY
A growing body of convergent data demonstrated hES cell neural retina commitment potential after embryoid body formation, and capacity to further differentiate into cells expressing photoreceptor markers (Zhong etal., 2014; Meyer etal., 2009). The different methods previously developed, though real advances, still suffer from drawbacks generally associated with pluripotent stem cell differentiation into highly specialized cell types. These protocols for photoreceptor-directed differentiation of hES or hiPS cells require several steps, addition of several molecules, and are rather inefficient. Current methods for differentiation into the photoreceptor cell lineage require the formation of embryoid bodies and/or require manual selection of retinal cells from culture. Thus, there is a need in the art for efficient and reliable large-scale methods for obtaining substantially pure cultures of certain human neuroepithelial lineage cells, particularly photoreceptor precursor (PRP) cells.
SUMMARY
[0006] The present embodiments provide methods and compositions for the production of ocular cells and use thereof In a first embodiment, there are provided methods for producing a population of neural retinal progenitors (NRPs) comprising obtaining a starting population of human induced pluripotent stem cells (iPSCs); culturing the iPSCs in retinal induction medium (RIM) to initiate differentiation of the cells into anterior neuroectoderm cells; further culturing the cells in a first retinal differentiation medium (RD1) comprising a BMP
inhibitor to further differentiate the cells to anterior neuroectoderm cells; inducing retinal differentiation of the anterior neuroectoderm cells by culturing the cells in a second retinal differentiation medium (RD2) essentially free of BMP inhibitors to form retinal progenitor cells (RPCs); and culturing the RPCs in a retinal maturation (RM) medium to produce NRPs. In a further embodiment, there are provided methods for producing a population of NRPs comprising obtaining a starting population of human iPSCs; culturing the cells in RD1 comprising a BMP
inhibitor to further differentiate the cells to anterior neuroectoderm cells; inducing retinal differentiation of the anterior neuroectoderm cells by culturing the cells in RD2 essentially free of BMP inhibitors to form RPCs; and culturing the RPCs in RM medium to produce NRPs. In particular aspects, the culturing is further defined as adherent 2-dimensional culture.
inhibitor to further differentiate the cells to anterior neuroectoderm cells; inducing retinal differentiation of the anterior neuroectoderm cells by culturing the cells in a second retinal differentiation medium (RD2) essentially free of BMP inhibitors to form retinal progenitor cells (RPCs); and culturing the RPCs in a retinal maturation (RM) medium to produce NRPs. In a further embodiment, there are provided methods for producing a population of NRPs comprising obtaining a starting population of human iPSCs; culturing the cells in RD1 comprising a BMP
inhibitor to further differentiate the cells to anterior neuroectoderm cells; inducing retinal differentiation of the anterior neuroectoderm cells by culturing the cells in RD2 essentially free of BMP inhibitors to form RPCs; and culturing the RPCs in RM medium to produce NRPs. In particular aspects, the culturing is further defined as adherent 2-dimensional culture.
[0007] In some aspects, the method further comprises culturing the iPSCs in retinal induction medium (RIM) to initiate differentiation of the cells into anterior neuroectoderm cells prior to culturing the cells in RD1. In certain aspects, the method further comprises culturing the population of NRPs as suspension aggregates in medium comprising a y-secretase inhibitor and a ROCK inhibitor.
[0008] In some aspects of the above embodiments, the methods further comprise culturing the NRPs in FDSC medium comprising a y-secretase inhibitor and FGF
for a period of time sufficient to produce a population of NRPs.
for a period of time sufficient to produce a population of NRPs.
[0009] In certain aspects, the RIM comprises a BMP inhibitor, a TGFr3 inhibitor, and/or IGF-1. In certain aspects, the RIM comprises a BMP inhibitor, a TGFr3 inhibitor, a Wnt inhibitor, and/or IGF-1. In particular aspects, the RIM is essentially free of activin A. In particular aspects, the RIM is essentially free of a Wnt inhibitor, such as CKI-7. In some aspects, culturing the iPSCs in RIM is for 1-3 days, such as 1, 2, or 3 days.
[0010] In some aspects, the RD1 medium further comprises a TGFr3 inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain aspects, the RD1 medium further comprises a TGFr3 inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain aspects, the RD1 media does not comprise CKI-7. In some aspects, the culturing in RD1 is for 1-3 days, such as 1, 2, or 3 days.
[0011] In some aspects, the RD2 medium comprises a TGFr3 inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain aspects, an increase in VSX2 expression of the anterior neuroectoderm cells is measured for differentiation potential. In particular aspects, the RD2 medium is essentially free of LDN193189. In some aspects, culturing in RD2 is for 5-9 days, such as 5, 6, 7, 8, or 9 days. In certain aspects, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells after culturing of in RD2 express PMEL17.
In certain aspects, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells after culturing in RD2 express VSX2.
In particular aspects, the RPCs express PAX6, MITF, and/or PMEL. In some aspects, the RPCs do not express or have essentially no expression of TRYP1, CRALBP, and/or BEST1.
In certain aspects, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells after culturing in RD2 express VSX2.
In particular aspects, the RPCs express PAX6, MITF, and/or PMEL. In some aspects, the RPCs do not express or have essentially no expression of TRYP1, CRALBP, and/or BEST1.
[0012] In some aspects, the culturing from obtaining iPSCs to culturing in RM
is further defined as adherent 2-dimensional culture. In some aspects, the culturing from obtaining iPSCs to culturing in RM is essentially free of aggregates.
is further defined as adherent 2-dimensional culture. In some aspects, the culturing from obtaining iPSCs to culturing in RM is essentially free of aggregates.
[0013] In some aspects, the RM media comprises nicotinamide and ascorbic acid.
In certain aspects, the RM medium further comprises FGF and a TGFr3 inhibitor. In some aspects, the RM medium further comprises FGF, SB431542, CKI-7, and IGF-1. In some aspects, the RM medium further comprises a y-secretase inhibitor. In certain aspects, the culturing in RM
medium to produce NRPs is for 3-7 days. In some aspects, the NRPs express PAX6 and VSX2.
In certain aspects, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells after culturing in RM medium to produce NRPs express PAX6 and VSX2. In particular aspects, the RPCs express PAX6, MITF, and/or PMEL.
In certain aspects, the RM medium further comprises FGF and a TGFr3 inhibitor. In some aspects, the RM medium further comprises FGF, SB431542, CKI-7, and IGF-1. In some aspects, the RM medium further comprises a y-secretase inhibitor. In certain aspects, the culturing in RM
medium to produce NRPs is for 3-7 days. In some aspects, the NRPs express PAX6 and VSX2.
In certain aspects, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells after culturing in RM medium to produce NRPs express PAX6 and VSX2. In particular aspects, the RPCs express PAX6, MITF, and/or PMEL.
[0014] In another embodiment, there are provided methods for producing a population of photoreceptor precursor cells (PRPs) comprising obtaining a starting population of NRPs according to the embodiments; and further culturing the NRPs in photoreceptor precursor induction medium (FDSC) medium comprising a y-secretase inhibitor and FGF for a period of time sufficient to produce a population of PRPs. In some aspects, the culturing is further defined as adherent 2-dimensional culture.
[0015] In some aspects, the culturing of the NRPs in FDSC medium comprising a y-secretase inhibitor and FGF for a period of time sufficient to produce a population of PRPs is for 10-20, 20-30, 30-40, or 40-50 days, such as 11, 12, 13, 14, 15, 16, 17, 18, or 19 days or more. the FDSC further comprises a TGF13 inhibitor and WNT inhibitor. In some aspects, the RM medium further comprises FGF and a TGFI3 inhibitor.
[0016] In some aspects, the method to produce PRPs further comprises maturing the population of PRPs as suspension aggregates in RM medium or a photoreceptor maturation (PM) medium comprising nicotinamide, thereby producing a population of mature PRP
aggregates. In some aspects, maturing is for 6-10 days, such as 6, 7, 8, 9, or 10 days. In additional aspects, the method further comprises cryopreserving the mature PRP
aggregates.
In some aspects, the PM medium further comprises a y-secretase inhibitor.
aggregates. In some aspects, maturing is for 6-10 days, such as 6, 7, 8, 9, or 10 days. In additional aspects, the method further comprises cryopreserving the mature PRP
aggregates.
In some aspects, the PM medium further comprises a y-secretase inhibitor.
[0017] In certain aspects, the method to produce PRPs further comprises dissociating the mature PRP aggregates into essentially single cells in PM medium. In some aspects, the method comprises cryopreserving the mature PRPs as single cells.
[0018] In some aspects, the PRPs are cultured as adherent cells in PM medium for 5-12 days, such as 5, 6, 7, 8, 9, 10, 11, or 12 days. In certain aspects, the PM
medium further comprises a y-secretase inhibitor. In certain aspects, the dissociated cells are re-aggregated. In some aspects, the PM medium further comprises a CDK inhibitor. In particular aspects, at least 750o, 760o, 770o, 780o, 790o, 800o, 810o, 820o, 830o, 840o, 850o, 900o, 950o, or 10000 of the cells express Recoverin (RCVRN).
medium further comprises a y-secretase inhibitor. In certain aspects, the dissociated cells are re-aggregated. In some aspects, the PM medium further comprises a CDK inhibitor. In particular aspects, at least 750o, 760o, 770o, 780o, 790o, 800o, 810o, 820o, 830o, 840o, 850o, 900o, 950o, or 10000 of the cells express Recoverin (RCVRN).
[0019] In some aspects, the method further comprises purifying the PRPs. In certain aspects, purifying comprises selecting cells that are positive for CD171, thereby providing a purified PRP cell population. In some aspects, purifying comprises selecting cells that are positive for CD171 and/or SUSD2, thereby providing a purified PRP cell population. In particular aspects, purifying comprises selecting cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, thereby providing a purified PRP cell population. In specific aspects, purifying comprises selecting cells that are positive for CD111, CD133, CD230, and/or CD344, thereby providing a purified PRP cell population. In some aspects, purifying comprises selecting cells that are positive for CD344, thereby providing a purified PRP cell population. In some aspects, purifying is performed at Day 65 or Day 75. In certain aspects, purifying comprises depletion of cells positive for two or more of the markers selected from the group consisting of CD9, CD49f, CD340, podoplanin, CD29, CD63, and CD298.
[0020] In some aspects, at least 900o, 910o, 92%, 930o, 940o, 950o, 96%, 970o, 98%, 99%, or 100% of the cells in the purified PRP cell population express Class III 0-tubulin (TUBB3). In certain aspects, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, .. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the purified PRP cell population express RCVRN. In some aspects, at least 700o, 710o, 72%, 730o, 740o, 750o, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the purified PRP
cell population express RCVRN. In some aspects, the PRPs express one or more markers selected from the group consisting of OTX2, CRX, BLIMP1, NEUROD1, RCVRN, TUBB3 and CD171/L1CAM. In certain aspects, the PRPs do not express or have essentially no expression of TRYP1, CRALBP, BEST1, Ki67, MITF, and/or PMEL17. In some aspects, the cells have low or essentially no expression of PAX6, CHX10 (also referred to herein as VSX2; both terms are used interchangeably herein) and/or Onecutl. In particular aspects, less than 150o, 10%, or 5% (e.g., less than 4%, 3%, 2%, or 1%) of the cells in the purified PRP
population express PAX6.
cell population express RCVRN. In some aspects, the PRPs express one or more markers selected from the group consisting of OTX2, CRX, BLIMP1, NEUROD1, RCVRN, TUBB3 and CD171/L1CAM. In certain aspects, the PRPs do not express or have essentially no expression of TRYP1, CRALBP, BEST1, Ki67, MITF, and/or PMEL17. In some aspects, the cells have low or essentially no expression of PAX6, CHX10 (also referred to herein as VSX2; both terms are used interchangeably herein) and/or Onecutl. In particular aspects, less than 150o, 10%, or 5% (e.g., less than 4%, 3%, 2%, or 1%) of the cells in the purified PRP
population express PAX6.
[0021] A further embodiment provides methods for producing a population of PRPs comprising obtaining a starting population of NRPs according to the embodiments; and further culturing the NRPs in PRP maturation medium (PM) comprising a cyclin-dependent kinase inhibitor for a period of time sufficient to produce a population of PRPs.
[0022] In some aspects, at least 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100%) of the cells are positive for PAX6 and prior to culturing in PM medium. In certain aspects, the NRPs are cultured as aggregates in the presence of a y-secretase inhibitor and a ROCK inhibitor. In some aspects, the PM further comprises a y-secretase inhibitor. In particular aspects, the PM further comprises a MEK
inhibitor.
inhibitor.
[0023] In additional aspects, the method further comprising dissociating the PRPs into essentially single cells in PM medium. In some aspects, the PRPs are cultured as adherent cells in PM medium.
[0024] In some aspects, the method further comprises enriching for PRPs by selecting for cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, and/or removing cells that are positive for CD9, CD49f, CD340, podoplanin CD29, CD63, and/or CD298. In some aspects, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the cells are positive for TUBB3 and/or at least 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100%) of the cells are positive for RCVRN.
[0025] In yet another embodiment, there are provided methods for producing a population of optic vesicles (OVs) comprising obtaining a starting population of PRPs according to the embodiments; and further culturing the PRPs as suspension aggregates in RM
medium or PRP maturation (PM) medium for a period of time sufficient to produce a population of OVs.
medium or PRP maturation (PM) medium for a period of time sufficient to produce a population of OVs.
[0026] In some aspects, the culturing of PRPs as suspension aggregates in RM
medium or PRP maturation (PM) medium for a period of time sufficient to produce a population of OVs is for at least 20 days, such as at least, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more days. In some aspects, the RM medium further comprises a y-secretase inhibitor. In certain aspects, the RM medium further comprises FGF and SB431542. In some aspects, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the population of OVs express VSX2. In some aspects, at least 10%, 1100, 120o, 130o, 140o, 150o, 160o, 170o, 180o, 190o, 200o, 250o, 300o, 400o, 500o, 60% or higher of the cells in the population of OVs express RCVRN. In some aspects, the OVs express Gamma-synuclein (SNCG), Opsin, RCVRN, and Rhodopsin. In certain aspects, the FDSC further comprises a TGFr3 inhibitor and a Wnt inhibitor.
medium or PRP maturation (PM) medium for a period of time sufficient to produce a population of OVs is for at least 20 days, such as at least, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more days. In some aspects, the RM medium further comprises a y-secretase inhibitor. In certain aspects, the RM medium further comprises FGF and SB431542. In some aspects, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the population of OVs express VSX2. In some aspects, at least 10%, 1100, 120o, 130o, 140o, 150o, 160o, 170o, 180o, 190o, 200o, 250o, 300o, 400o, 500o, 60% or higher of the cells in the population of OVs express RCVRN. In some aspects, the OVs express Gamma-synuclein (SNCG), Opsin, RCVRN, and Rhodopsin. In certain aspects, the FDSC further comprises a TGFr3 inhibitor and a Wnt inhibitor.
[0027] In some aspects, the culturing of NRPs in RM medium comprising a y-secretase inhibitor and FGF for a period of time sufficient to produce a population of PRPs is for 10-20 days, such as 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or more days. In some aspects, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher of the cells in the population of PRPs express TUBB3. In some aspects, at least 300o, 310o, 32%, 330o, 340o, 350o, 36%, 370o, 38%, 390o, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells in the population of PRPs express RCVRN.
[0028] In additional aspects, the methods further comprise culturing the population of NRPs as suspension aggregates in medium comprising a y-secretase inhibitor and a ROCK
inhibitor. In some aspects, the medium further comprises ascorbic acid and nicotinamide. In certain aspects, at least 75%, 76%, 770o, 78%, 790o, 800o, 85%, 900o, 950o, or 1000o of the cells in the population of NRPs express VSX2.
inhibitor. In some aspects, the medium further comprises ascorbic acid and nicotinamide. In certain aspects, at least 75%, 76%, 770o, 78%, 790o, 800o, 85%, 900o, 950o, or 1000o of the cells in the population of NRPs express VSX2.
[0029] In another embodiments, there are provided pharmaceutical compositions comprising the PRPs produced according to the embodiments, the OVs produced according to the embodiments, or the NRPs produced according to the embodiments, and a pharmaceutically acceptable carrier.
[0030] A further embodiment provides methods of treating injury or degeneration of retinal neurons in a subject comprising administering an effective amount of the pharmaceutical composition of the embodiments (e.g., PRPs, OVs, or NRPs produced herein) to an eye of the subject. In some aspects, the retinal neurons are photoreceptors.
[0031] The ocular cells, such as PRP cells, produced by the methods herein may be used in any methods and applications currently known in the art for photoreceptor cells. For example, a method of assessing a compound may be provided, comprising assaying a pharmacological or toxicological property of the compound on the photoreceptor cell. There may also be provided a method of assessing a compound for an effect on a PRP
cell, comprising: a) contacting the PRP cells provided herein with the compound; and b) assaying an effect of the compound on the PRP cells.
100321 In certain aspects of the above embodiments, the RIM comprises LDN193189, SB431542, CKI-7, and IGF-1. In some aspects, the RD1 medium further comprises a TGFr3 inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain aspects, the RD2 medium comprises a TGF13 inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In some aspects, the BMP inhibitor is LDN193189. In some aspects, the TGF13 is further defined as a type I receptor activin receptor-like kinase (ALK5) inhibitor. In certain aspects, the TGF13 inhibitor is SB431542, 6SB525334, SB- 505124, Lefty, A 83-01, D 4476, GW 788388, LY 364847, R
268712, or RepSox. In some aspects, the WNT inhibitor is CKI-7, IWP2, IWP4, XAV939, TAK 715, DKK1, or SFRP1. In particular aspects, the MEK inhibitor is PD0325901.
In some aspects, RD2 medium comprises 5B431542, CKI-7, IGF-1, and PD0325901.
In specific aspects, the RM medium further comprises FGF, 5B431542, CKI-7, and IGF-1. In some aspects, the y-secretase inhibitor is DAPT, Begacestat, Compound W, JLK6, L-685,485, Flurizan, DBZ, MRK560, PF3084014 hydrobromide, or BM5299897. In some aspects, the cycling dependent kinase (CDK) inhibitor is a CDK4/6 inhibitor, such as (palbociclib).
[0033] In another embodiment, there is provided a composition comprising a NRP
population, wherein at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP population express PMEL17, and/or at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the NRP population express VSX2. In some aspects, at least 95% of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP population express PMEL17, and/or at least 75% of the cells in the NRP population express VSX2.
In certain aspects, at least 95% of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP
population express PMEL17, and at least 75% of the cells in the NRP population express VSX2. In some aspects, the cells in the NRP population further express Ki67.
[0034] In another embodiment, there is provided a composition comprising a PRP
population, wherein at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the PRP population express TUBB3, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and/or less than 15% of the cells in the PRP
population express PAX6. In some aspects, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP population express RCVRN. In certain aspects, less than 10% or 5% of the cells in the PRP
population express .. PAX6. In some aspects, at least 90% of the cells in the PRP population express TUBB3, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and less than 15% of the cells in the PRP population express PAX6. In some aspects, at least 90% of the cells in the PRP population express TUBB3, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and/or less than 5% of the cells in the PRP population express PAX6. In certain aspects, the PRPs express one or more markers selected from the group consisting of OTX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, RCVRN, TUBB3 and CD171/L1CAM.
In some aspects, the PRPs do not express or have essentially no expression of TRYP1, CRALBP, BEST1, 1(167, MITF, and/or PMEL17.
[0035] A further embodiment provides a composition comprising a population of OVs, wherein at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells express VSX2 and/or at least 20%
of the cells express RCVRN. In some aspects, at least 65% of the cells express VSX2 and/or at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells express RCVRN. In certain aspects, at least 65%
of the cells express VSX2 and at least 30% of the cells express RCVRN.
[0036] Another embodiment provides a method for providing an enriched population of PRP cells comprising obtaining a starting cell population comprising PRP
cells; and enriching said starting cell population for PRP cells by selecting for cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, and/or removing therefrom cells that are positive for CD9, CD49f, CD340, podoplanin, CD29, CD63, and/or CD298, thereby providing a PRP-enriched cell population that is enriched for PRP cells as compared to the starting cell population. In some aspects, the method further comprises determining a level of enrichment of the PRP cells in said PRP-enriched population.
In some aspects, the level of enrichment is determined through the use of a cell marker selected from the group consisting of TUBB3, RCVRN, OTX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, and CD171/L1CAM. In certain aspects, the PRP-enriched cell population is enriched for PRP cells as compared to the starting cell population as determined by RCVRN
sorting. In some aspects, the PRP-enriched population is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% PRP
cells.
In certain aspects, the PRP-enriched population is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% PRP cells. In some aspects, the PRP-enriched population is essentially pure PRP cells. In particular aspects, the PRP cells are human PRP cells. In some aspects, the starting cell population is prepared from iPSCs. In some .. aspects, selecting and/or removing is performed by magnetic bead-based sorting or fluorescence-based sorting. In some aspects, the method comprises selecting for cells that are positive for CD171, SUSD2, CD111, CD133, CD230, and/or CD344.
[0037] Another embodiment provides a method for performing quality control during the production of an NRP cell product comprising detecting the expression of cell markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL. In some aspects, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells are positive for PAX6, CHX10 (VSX2), Ki67, and PMEL.
In certain aspects, at least 90% of the cells are positive for PAX6, CHX10 (VSX2), Ki67, and PMEL.
[0038] In a further embodiment, there is provided a method for performing quality .. control during the production of a PRP cell product comprising detecting the expression of off-target cell markers selected from the group consisting of PAX6, ONECUT1, HNCHF6, CHX10, and Ki67. In some aspects, the off-target cells are positive for PAX6 and ISL1. In some aspects, a PRP cell product passing quality control comprises less than 10% or 5% PAX6-positive cells. In certain aspects, a PRP cell product passing quality control comprises less than 10% PAX6-positive cells, less than 0.05% Ki67-positive cells, less than 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100%
CHX10-positive cells, and/or less than 2% ONECUT1-positive cells. In some aspects, a PRP
cell product passing quality control comprises less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15% CHX10-positive cells, and/or less than 1%
ONECUT1-positive cells. In certain aspects, a PRP cell product passing quality control comprises less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15% CHX10-positive cells, and less than 1% ONECUT1-positive cells.
[0039] A further embodiment provides a method for performing quality control during the production of an OV cell product comprising detecting the expression of one or more cell markers selected from the group consisting of RCVRN, CHX10, PAX6, and Ki67. In certain aspects, the cells markers are RCVRN and CHX10. In particular aspects, at least 60% of the cells in the OV cell product are positive for RCVRN and at least 30%, 31%,
cell, comprising: a) contacting the PRP cells provided herein with the compound; and b) assaying an effect of the compound on the PRP cells.
100321 In certain aspects of the above embodiments, the RIM comprises LDN193189, SB431542, CKI-7, and IGF-1. In some aspects, the RD1 medium further comprises a TGFr3 inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain aspects, the RD2 medium comprises a TGF13 inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In some aspects, the BMP inhibitor is LDN193189. In some aspects, the TGF13 is further defined as a type I receptor activin receptor-like kinase (ALK5) inhibitor. In certain aspects, the TGF13 inhibitor is SB431542, 6SB525334, SB- 505124, Lefty, A 83-01, D 4476, GW 788388, LY 364847, R
268712, or RepSox. In some aspects, the WNT inhibitor is CKI-7, IWP2, IWP4, XAV939, TAK 715, DKK1, or SFRP1. In particular aspects, the MEK inhibitor is PD0325901.
In some aspects, RD2 medium comprises 5B431542, CKI-7, IGF-1, and PD0325901.
In specific aspects, the RM medium further comprises FGF, 5B431542, CKI-7, and IGF-1. In some aspects, the y-secretase inhibitor is DAPT, Begacestat, Compound W, JLK6, L-685,485, Flurizan, DBZ, MRK560, PF3084014 hydrobromide, or BM5299897. In some aspects, the cycling dependent kinase (CDK) inhibitor is a CDK4/6 inhibitor, such as (palbociclib).
[0033] In another embodiment, there is provided a composition comprising a NRP
population, wherein at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP population express PMEL17, and/or at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the NRP population express VSX2. In some aspects, at least 95% of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP population express PMEL17, and/or at least 75% of the cells in the NRP population express VSX2.
In certain aspects, at least 95% of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the NRP
population express PMEL17, and at least 75% of the cells in the NRP population express VSX2. In some aspects, the cells in the NRP population further express Ki67.
[0034] In another embodiment, there is provided a composition comprising a PRP
population, wherein at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells in the PRP population express TUBB3, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and/or less than 15% of the cells in the PRP
population express PAX6. In some aspects, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP population express RCVRN. In certain aspects, less than 10% or 5% of the cells in the PRP
population express .. PAX6. In some aspects, at least 90% of the cells in the PRP population express TUBB3, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and less than 15% of the cells in the PRP population express PAX6. In some aspects, at least 90% of the cells in the PRP population express TUBB3, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and/or less than 5% of the cells in the PRP population express PAX6. In certain aspects, the PRPs express one or more markers selected from the group consisting of OTX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, RCVRN, TUBB3 and CD171/L1CAM.
In some aspects, the PRPs do not express or have essentially no expression of TRYP1, CRALBP, BEST1, 1(167, MITF, and/or PMEL17.
[0035] A further embodiment provides a composition comprising a population of OVs, wherein at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells express VSX2 and/or at least 20%
of the cells express RCVRN. In some aspects, at least 65% of the cells express VSX2 and/or at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells express RCVRN. In certain aspects, at least 65%
of the cells express VSX2 and at least 30% of the cells express RCVRN.
[0036] Another embodiment provides a method for providing an enriched population of PRP cells comprising obtaining a starting cell population comprising PRP
cells; and enriching said starting cell population for PRP cells by selecting for cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, and/or removing therefrom cells that are positive for CD9, CD49f, CD340, podoplanin, CD29, CD63, and/or CD298, thereby providing a PRP-enriched cell population that is enriched for PRP cells as compared to the starting cell population. In some aspects, the method further comprises determining a level of enrichment of the PRP cells in said PRP-enriched population.
In some aspects, the level of enrichment is determined through the use of a cell marker selected from the group consisting of TUBB3, RCVRN, OTX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, and CD171/L1CAM. In certain aspects, the PRP-enriched cell population is enriched for PRP cells as compared to the starting cell population as determined by RCVRN
sorting. In some aspects, the PRP-enriched population is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% PRP
cells.
In certain aspects, the PRP-enriched population is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% PRP cells. In some aspects, the PRP-enriched population is essentially pure PRP cells. In particular aspects, the PRP cells are human PRP cells. In some aspects, the starting cell population is prepared from iPSCs. In some .. aspects, selecting and/or removing is performed by magnetic bead-based sorting or fluorescence-based sorting. In some aspects, the method comprises selecting for cells that are positive for CD171, SUSD2, CD111, CD133, CD230, and/or CD344.
[0037] Another embodiment provides a method for performing quality control during the production of an NRP cell product comprising detecting the expression of cell markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL. In some aspects, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells are positive for PAX6, CHX10 (VSX2), Ki67, and PMEL.
In certain aspects, at least 90% of the cells are positive for PAX6, CHX10 (VSX2), Ki67, and PMEL.
[0038] In a further embodiment, there is provided a method for performing quality .. control during the production of a PRP cell product comprising detecting the expression of off-target cell markers selected from the group consisting of PAX6, ONECUT1, HNCHF6, CHX10, and Ki67. In some aspects, the off-target cells are positive for PAX6 and ISL1. In some aspects, a PRP cell product passing quality control comprises less than 10% or 5% PAX6-positive cells. In certain aspects, a PRP cell product passing quality control comprises less than 10% PAX6-positive cells, less than 0.05% Ki67-positive cells, less than 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100%
CHX10-positive cells, and/or less than 2% ONECUT1-positive cells. In some aspects, a PRP
cell product passing quality control comprises less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15% CHX10-positive cells, and/or less than 1%
ONECUT1-positive cells. In certain aspects, a PRP cell product passing quality control comprises less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15% CHX10-positive cells, and less than 1% ONECUT1-positive cells.
[0039] A further embodiment provides a method for performing quality control during the production of an OV cell product comprising detecting the expression of one or more cell markers selected from the group consisting of RCVRN, CHX10, PAX6, and Ki67. In certain aspects, the cells markers are RCVRN and CHX10. In particular aspects, at least 60% of the cells in the OV cell product are positive for RCVRN and at least 30%, 31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100%
of the cells in the OV cell product are positive for CHX10. In some aspects, the cell markers are PAX6 and Ki67. In some aspects, the method further comprises detecting the absence of TYRP1. In some aspects, the method further comprises detecting the expression of one or more markers selected from the group consisting of MITF, CRALBP, BEST1, OTX2, CRX, BLIMP1, NEUROD1, TUBB3, ONECUT1, and CD171/L1CAM.
[0040] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0042] FIG. 1: Candidate Overview. Schematic of iPSC-derived ocular cell differentiation including neural retinal progenitor cells (NRPs), photoreceptor precursor cells (PRPs) and optic vesicles (0Vs). Candidate 1 (C-1) represents PRP
differentiated under adherent 2-dimensional (2-D) conditions (2-D PRP). Candidate 2 (C-2) represents PRP
differentiated under both 2-D and suspension aggregate 3-dimensional (3-D) conditions (Hybrid PRP). Candidate 3 (C-3) represents OVs differentiated under both 2-D
and 3-D
conditions (Hybrid OV). Candidate 4 (C-4) represents NRPs differentiated under conditions.
[0043] FIG. 2: Schematic of the 2-D PRP differentiation process including media phase duration.
[0044] FIGS. 3A-3C: 2-D PRP MACS Enrichment. Flow cytometry analysis of 2-D
PRP for fIII-tubulin (TUBB3) and Recoverin (RCVRN) expression both (FIG. 3A) pre- and (FIG. 3B) post-MACS enrichment. (FIG. 3C) Pre- and post-MACS flow cytometry analysis summary for 2-D PRP differentiations (n=5).
[0045] FIGS. 4A-4D: 2-D PRP Characterization. (FIG. 4A) Image of day 2 post-thaw 2-D PRP aggregates (10x objective). (FIG. 4B) Immunofluorescence staining of day 1 post-thaw 2-D PRPs for TUBB3 and RCVRN (20x objective). (FIG. 4C) Immunofluorescence staining of day 1 post-thaw 2-D PRPs for CRX and RCVRN (20x objective). (FIG.
4D) Single cell gene expression analysis of day 2 post-thaw 2-D PRP aggregates.
[0046] FIG. 5: Schematic of the Hybrid PRP differentiation process.
[0047] FIG. 6: Input iPSC Density Optimization. Flow cytometry analysis of day Hybrid PRP differentiation for Pax6 and Vsx2 with input densities ranging from 1x104 (10K) to 1x106 (1000K) cells per well of a 6 well plate.
[0048] FIG. 7: RM Medium Optimization. Flow cytometry analysis of day 18 RPCs grown in RM medium with or without the addition of FGF2 and SB431542.
[0049] FIGS. 8A-8B: PM Medium Optimization. (FIG. 8A) Morphology of late-stage plated PRP in PM medium with or without the addition of PD0332991 or Activin A. (FIG. 8B) Flow cytometry analysis of late-stage plated C-2 PRP in PM medium with or without the addition of PD0332991 or Activin A.
[0050] FIG. 9: Hybrid PRP MACS Enrichment. Flow cytometry analysis of Hybrid PRP for TUBB3, NESTIN and RCVRN expression both pre- and post-MACS enrichment.
[0051] FIGS. 10A-10D: Hybrid PRP Characterization. (FIG. 10A) Image of day 2 .. post-thaw Hybrid PRP aggregates (10x objective). (FIG. 10B) Immunofluorescence staining of day 1 post-thaw Hybrid PRP for TUBB3 and RCVRN (20x objective). (FIG. 10C) Immunofluorescence staining of day 1 post-thaw Hybrid PRP for CRX and RCVRN
(20x objective). (FIG. 10D). Single cell gene expression analysis of day 2 post-thaw Hybrid PRP
aggregates.
[0052] FIG. 11: Schematic of the Hybrid OV differentiation process.
[0053] FIGS. 12A-12D: Hybrid OV Morphology and Characterization. (FIG. 12A) Image of day 68 differentiating Hybrid OV. (FIG. 12B) Immunofluorescence staining time course of differentiating Hybrid OV for VSX2 and RCVRN. (FIG 12C) Immunofluorescence staining of late-stage Hybrid OV for Gamma-synuclein (SNCG; retinal ganglion cell marker), RCVRN (PRP and photoreceptor marker), Green-sensitive Opsin (OPN1MW; cone marker), cone-arrestin (ARR3; cone marker) and Rhodopsin (RHO; rod marker). (FIG 12D) Flow cytometry time course analysis of differentiating Hybrid OV for VSX2 and RCVRN.
[0054] FIG. 13: Schematic of the 2-D NRP differentiation process.
[0055] FIGS. 14A-14C: 2-D NRP Characterization. (FIG. 14A) Image of day 2 post-thaw NRP aggregates. (FIG. 14B) Immunofluorescence staining of day 1 post-thaw adherent NRP for PAX6 (left) and VSX2 (right). (FIG. 14C) Flow cytometry analysis of day 2 post-thaw NRP for PAX6, PMEL17 and VSX2.
[0056] FIGS. 15A-15B: Flow analyses of surface antigens and recoverin co-expression corresponding to Table 4 was determined using a quadrant flow plot following dual-labeling with recoverin and the antibody against the respective surface antigen.
Additionally, percent population of cells expressing only recoverin (in FITC, x-axis) or only the surface antigen (in APC, y-axis) was evaluated. All plots were gated against unstained (empty) cells and the corresponding isotype control (REA IgGl, MsIgGl, Ms IgG2a, MsIgG2b, MsIgM).
[0057] FIG. 16: Time course of recoverin expression following enrichment with surface antigens. Recoverin expression is greatest at D65 for CD344-enriched PRP.
Enrichment with CD111 and CD230 also yields high percentage of recoverin expression, compared to pre-enriched (pre-MACS) cells.
[0058] FIG. 17: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with surface antigens CD111, CD230 and CD344 at D55.
[0059] FIG. 18: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with surface antigens CD111, CD230 and CD344 at D65.
[0060] FIG. 19: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with surface antigens CD111, CD230 and CD344 at D75.
[0061] FIG. 20: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with CD133 at D75.
[0062] FIG. 21: Flow analyses of depletion markers.
[0063] FIG. 22: Flow cytometry analysis of PAX6 and CHX10 expression with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
[0064] FIG. 23: Percent expression of PAX6 and CHX10 with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
[0065] FIGS. 24A-24B: (FIG. 24A) Flow cytometry analysis of Ki67 and CHX10 expression with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
*Using conjugated CHX10 shifted the plot (higher) but in this case reduced the expression level; it may be possible that the antibody did not label maximum epitopes due to steric hindrance.
(FIG. 24B) Percent expression of Ki67 and CHX10 with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
[0066] FIGS. 25A-25B: (FIG. 25A) Flow cytometry analysis of TYRP1 and PMEL
expression with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
(FIG. 25B) Percent expression of TYRP1 and PMEL with or without CKI-7 at Day 15 and Day 25 of PRP
differentiation.
[0067] FIGS. 26A-26B: Comparison of early eye field and RPE markers in the presence (FIG. 26A) and absence (FIG. 26B) of RIM.
[0068] FIGS. 27A-27D: Effects of RIM versus No RIM on early (D15) neural retina differentiation across lines on expression of PAX6 (FIG. 27A), CHX10 (FIG.
27B), TRYP10 (FIG. 27C), and PMEL (FIG. 27D).
[0069] FIGS. 28A-28D: Comparison of mid-stage differentiation (--D30) of PRP
in the presence or absence of RIM on expression of PAX6 (FIG. 28A), CHX10 (FIG. 28B), (FIG. 28C), and PMEL (FIG. 28D).
[0070] FIGS. 29A-29B: Expression of on-target PRP characterization markers at Day 75 in the presence (FIG. 29A) and absence (FIG. 29B) of RIM.
[0071] FIG. 30: Expression of off-target retinal cell markers at D75 differentiation in the presence or absence of RIM.
[0072] FIGS. 31A-31B: Recoverin expression is greater than 90% in PRP cells enriched with SUSD2 compared to CD171. (FIG. 31A) SUSD2-enriched RCVRN-positive population. (FIG. 31B) CD171-enriched RCVRN-positive population.
[0073] FIGS. 32A-32E: Maximum recoverin (FIG. 32A) and SUSD2 (FIG. 32B) expression was observed between differentiation day D55 ¨ D65, with recoverin peaking soon after SUSD2. Recoverin and SUSD2 co-expression (FIG. 32C) mimics the SUSD2 alone expression, suggesting that SUSD2-expressing cells also express recoverin while not all recoverin-positive cells express SUSD2. An overlay of the three graphs clearly demonstrates the parallel expression of the dual recoverin/SUSD2 versus the single SUSD2 stain (FIG. 32D).
SUSD2-labeling with post-fixed recoverin labeling. Independent time course studies of recoverin and SUSD2 co-expression from early stage D55 through later stage D105 (FIG. 32E) supports peak SUSD2 expression around D65 but also shows equally elevated expression at D55.
[0074] FIGS. 33A-33C: Percentage recoverin expression increased further post SUSD2-enrichement at D65, D75 and D85 (FIG. 33A), showing highest expression at D65 which also corresponds to early peak SUSD2 and recoverin expression times.
Although percent recoverin-positive cell population decreases from D65 to D75 to D85 (B), SUSD2-enrichment still enhances percentage recoverin-positive cells compared to CD171-enrichment at D85 (FIG. 33B). However, a limitation of SUSD2-enrichment is low cell output, with <25%
of the initial cell input recovered following enrichment (FIG. 33C). This holds true for cells enriched with either SUSD2 or CD171 at D85, whereby only 3% of total input cells are enriched with SUSD2 while only 18% of total input cells are enriched by CD171.
The flow-through (labeled as SUSD2- and CD171-) appeared to contain the majority of the cells, suggesting a possible reduction in expression of both surface markers at later differentiation stages.
[0075] FIGS. 34A-34E: At D65, the height pre-MACS SUSD2 and pre- and post-MACS recoverin expression, CHX10 expression is at its lowest but significantly increases by D75 and D85 (FIG. 34A). Co-expression of recoverin-CHX10 was <3% at D65, rising and falling at D75 and D85, respectively, suggesting a short-lived bipolar cell population (FIG.
34B). NeuroD1 expression was highest at D65 pre- and post-enrichment compared to D75 and D85 (FIG. 34C) and NeuroD1 co-expression was also greatest at D65 and further enhanced post-SUSD2 enrichment (FIG. 34D). An additional experiment (FIG. 34E) confirmed low CHX10 at D55, and also showed substantial reduction in CHX10 after SUSD2 MACS
enrichment at D75.
I. Description of Illustrative Embodiments [0076] In certain embodiments, the present disclosure provides methods for producing ocular cells, including a photoreceptor precursor (PRP) cell population. PRP
cells can be derived from pluripotent stem cells such as ES cells or iPS cells in a defined 2D cell culture without the need for formation of embryoid bodies or selecting colonies of cells. Alternatively, the PRP cells may be derived in a hybrid adherent 2-D and suspension aggregate 3-D culture.
Briefly, the PSCs may be differentiated to anterior neuroectoderm cells which are cultured in a retinal differentiation media comprising a BMP inhibitor for a short period and then further cultured in a retinal differentiation media without the BMP inhibitor.
Interestingly, the inventors found that the removal of the BMP inhibitor enhances the neural retinal differentiation potential of the anterior neuroectoderm cells. The cells may then be further differentiated into retinal progenitor cells (RPCs) in the presence of nicotinamide and the absence of Activin A to direct the cells toward a photoreceptor lineage instead of other retinal lineages, such as retinal pigment epithelial cells. Finally, the RPCs, may be differentiated in the presence of a TGFP inhibitor, a WNT inhibitor, basic FGF, and a y-secretase inhibitor to produce neural retinal progenitor (NRP) cells which may then be differentiated to PRP cells.
Thus, the present disclosure provides a highly efficient and reproducible method of differentiating PSCs into PRP cells.
[0077] The present disclosure also provides methods for the production of PRP
cells or optic vesicles through the combination of a 2D and 3D aggregate culture. The RPCs may be cultured as aggregates in retinal maturation media free of Activin A for a period of time to produce PRP aggregates which may then be dissociated and cultured as a monolayer.
Alternatively, the RPCs may be cultured in the retinal maturation media free of Activin A for an extended period of time to produce the PRP aggregates and eventually produce optic vesicles.
[0078] Further embodiments of the present disclosure provide a method of purifying the population of PRP cells that are obtained by the above methods. The purification method can comprise positive and/or negative selection. For example, cells which express CD171 may be selected for by cell sorting. Therefore, the purification process yields a PRP-enriched cell population that has a greater percentage of PRP cells than the population obtained after differentiation from the RPCs.
[0079] Thus, the present methods are more time- and cost-efficient, and may enable manufacture of PRP-enriched cell populations for therapeutics from a renewable source, stem cells, at a clinical scale. They may be used to uncover mechanisms, new genes, soluble or membrane-bound factors that are important for the development, differentiation, maintenance, survival and function of photoreceptor cells.
[0080] The PRP cells and photoreceptor cells provided herein may be used in a variety of in vivo and in vitro methods. For example, the PRP cells may be used in vivo to treat conditions of the retina, including but not limited to macular degeneration and retinitis pigmentosa. The PRP cells and photoreceptor cells may also be used in vitro in screening assays to identify putative therapeutic or prophylactic treatment candidates. Further embodiments and advantages of the present disclosure are described below.
I. Definitions [0081] The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, a purified population of cells is greater than about 90%
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, or, most preferably, essentially free of other cell types.
[0082] As used herein, "essentially" or "essentially free," in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0083] As used herein in the specification, "a" or "an" may mean one or more.
As used herein in the claim(s), when used in conjunction with the word "comprising,"
the words "a" or "an" may mean one or more than one.
[0084] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.
[0085] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0086] The term "cell population" is used herein to refer to a group of cells, typically of a common type. The cell population can be derived from a common progenitor or may comprise more than one cell type. An "enriched" cell population refers to a cell population derived from a starting cell population (e.g., an unfractionated, heterogeneous cell population) that contains a greater percentage of a specific cell type than the percentage of that cell type in the starting population. The cell populations may be enriched for one or more cell types and depleted of one or more cell types.
[0087] The term "stem cell" refers herein to a cell that under suitable conditions is capable of differentiating into a diverse range of specialized cell types, while under other suitable conditions is capable of self-renewing and remaining in an essentially undifferentiated pluripotent state. The term "stem cell" also encompasses a pluripotent cell, multipotent cell, precursor cell and progenitor cell. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from genital tissue of a fetus. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state by the expression of certain transcription factors associated with pluripotency; these cells are called "induced pluripotent stem cells" or "iPSCs".
[0088] The term "pluripotent" refers to the property of a cell to differentiate into all other cell types in an organism, with the exception of extraembryonic, or placental, cells.
Pluripotent stem cells are capable of differentiating to cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types) even after prolonged culture. A
pluripotent stem cell is an embryonic stem cell derived from the inner cell mass of a blastocyst.
In other embodiments, the pluripotent stem cell is an induced pluripotent stem cell derived by reprogramming somatic cells.
[0089] The term "differentiation" refers to the process by which an unspecialized cell becomes a more specialized type with changes in structural and/or functional properties. The mature cell typically has altered cellular structure and tissue-specific proteins.
[0090] As used herein, "undifferentiated" refers to cells that display characteristic markers and morphological characteristics of undifferentiated cells that clearly distinguish them from terminally differentiated cells of embryo or adult origin.
[0091] "Embryoid bodies (EBs)" are aggregates of pluripotent stem cells that can undergo differentiation into cells of the endoderm, mesoderm, and ectoderm germ layers. The spheroid structures form when pluripotent stem cells are allowed to aggregate under non-adherent culture conditions and thus form EBs in suspension.
[0092] An "isolated" cell has been substantially separated or purified from others cells in an organism or culture. Isolated cells can be, for example, at least 99%, at least 98% pure, at least 95% pure or at least 90% pure.
[0093] An "embryo" refers to a cellular mass obtained by one or more divisions of a zygote or an activated oocyte with an artificially reprogrammed nucleus.
[0094] An "embryonic stem (ES) cell" is an undifferentiated pluripotent cell which is obtained from an embryo in an early stage, such as the inner cell mass at the blastocyst stage, or produced by artificial means (e.g. nuclear transfer) and can give rise to any differentiated cell type in an embryo or an adult, including germ cells (e.g. sperm and eggs).
[0095] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming a somatic cell by expressing or inducing expression of a combination of factors (herein referred to as reprogramming factors). iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, 0ct4 (sometimes referred to as Oct 3/4), 5ox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram a somatic cell to a pluripotent stem cell.
[0096] An "allele" refers to one of two or more forms of a gene. Diploid organisms such as humans contain two copies of each chromosome, and thus carry one allele on each.
[0097] The term "homozygous" is defined as containing two of the same alleles at a particular locus. The term "heterozygous" refers to as containing two different alleles at a particular locus.
[0098] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. A haplotype can be based upon a set of single-nucleotide polymorphisms (SNPs) on a single chromosome and/or the alleles in the major histocompatibility complex.
[0099] As used herein, the term "haplotype-matched" is defined as the cell (e.g. iPS
cell) and the subject being treated share one or more major histocompatibility locus haplotypes.
The haplotype of the subject can be readily determined using assays well known in the art. The haplotype-matched iPS cell can be autologous or allogeneic. The autologous cells which are grown in tissue culture and differentiated to PRP cells inherently are haplotype-matched to the subject.
[00100]
"Substantially the same HLA type" indicates that the Human Leukocyte Antigen (HLA) type of donor matches with that of a patient to the extent that the transplanted cells, which have been obtained by inducing differentiation of iPSCs derived from the donor's somatic cells, can be engrafted when they are transplanted to the patient.
[00101]
"Super donors" are referred to herein as individuals that are homozygous for certain MHC class I and II genes. These homozygous individuals can serve as super donors and their cells, including tissues and other materials comprising their cells, can be transplanted in individuals that are either homozygous or heterozygous for that haplotype.
The super donor can be homozygous for the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ
locus/loci alleles, respectively.
[00102]
"Feeder-free" or "feeder-independent" is used herein to refer to a culture supplemented with cytokines and growth factors (e.g., TGFP, bFGF, LIF) as a replacement for the feeder cell layer. Thus, "feeder-free" or feeder-independent culture systems and media may be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state.
In some cases, feeder-free cultures utilize an animal-based matrix (e.g.
MATRIGELTm) or are grown on a substrate such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to remain in an essentially undifferentiated state without the need for mouse fibroblast "feeder layers."
[00103]
"Feeder layers" are defined herein as a coating layer of cells such as on the bottom of a culture dish. The feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.
[00104] The term "defined" or "fully-defined," when used in relation to a medium, an extracellular matrix, or a culture condition, refers to a medium, an extracellular matrix, or a culture condition in which the chemical composition and amounts of approximately all the components are known. For example, a defined medium does not contain undefined factors such as in fetal bovine serum, bovine serum albumin or human serum albumin.
Generally, a defined medium comprises a basal media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources) which is supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin.
An example of a fully defined medium is Essential 8TM medium.
[00105] For a medium, extracellular matrix, or culture system used with human cells, the term "Xeno-Free (XF)" refers to a condition in which the materials used are not of non-human animal-origin.
[00106]
"Pre-confluent" refers to a cell culture in which the proportion of the culture surface which is covered by cells is about 60-80%. Usually, pre-confluent refers to a culture in which about 70% of the culture surface is covered by cells.
[00107] The term "retinal progenitor cells", also called "retinal precursor cells"
or "RPCs", encompass cells which are competent for generating all cell types of the retina, including neural retina cells, such as rods, cones, photoreceptor precursor cells, as well as cells which can differentiate into RPE.
[00108] The term "neural retinal progenitors" or "NRPs" refers to cells which are restricted in their differentiation potential to neural retina cell types.
[00109] The terms "photoreceptor precursor cells" and "PRP" cells refer to cells differentiated from embryonic stem cells or induced pluripotent stem cells which can differentiate into photoreceptor cells that expresses the cell marker rhodopsin or any of the three cone opsins, and optionally express the rod or cone cGMP. The photoreceptors may be rod and/or cone photoreceptors.
[00110] The term "optic vesicles" or "OVs" refers to cell aggregates or organoids, including PRP cell aggregates, with a morphology comprising optic vesicle structures.
[00111]
"Retinal pigment epithelium" refers to a layer of pigmented cells between the choroid, a layer filled with blood vessels, and the neural retina.
[00112]
"Retinal Induction Medium (RIM)" refers herein to a growth media that comprises a WNT pathway inhibitor and a BMP pathway inhibitor and can result in the differentiation of PSCs to retinal lineage cells. The RIM also comprises a TGFr3 pathway inhibitor, and may comprise IGF-1 and ascorbic acid.
[00113] The "Retinal Differentiation Medium (RD)" is defined herein as a medium that comprises a WNT inhibitor, a TGFr3 inhibitor and a MEK inhibitor and differentiates anterior neuroectoderm cells. The RDM may (i.e., RD1) or may not (i.e., RD2) comprise a BMP inhibitor, and may comprise IGF-1 and ascorbic acid.
[00114] The "Retinal Maturation Medium (RM)" is defined as a growth medium for the culture of retinal cells comprising Nicotinamide and ascorbic acid.
The RM is preferably free of Activin A. The RM may (i.e., RM1) or may not (i.e., RM2) comprise a y secretase inhibitor, such as DAPT, or a TGFr3 inhibitor, such as SB431542, and may comprise IGF-1 and ascorbic acid.
[00115] The "PRP Maturation Medium (PM)" is referred to herein as a growth medium for the culture of PRP cells comprising Nicotinamide and a y secretase inhibitor, such as DAPT. The PM may (i.e., PM1) or may not (i.e., PM2) contain a CDK
inhibitor, such as PD0332291, a TGF-P pathway activator, such as Activin A, or a mitogen, such as retinoic acid.
[00116] The "Photoreceptor Precursor Induction Medium (FDSC)" refers to a growth medium which comprises a TGFr3 inhibitor, a WNT inhibitor, and a y-secretase inhibitor. The FDSC may comprise basic FGF and ascorbic acid.
[00117] The term "retinal degeneration-related disease" is intended to refer to any disease resulting from innate or postnatal retinal degeneration or abnormalities. Examples of retinal degeneration-related diseases include retinal dysplasia, retinal degeneration, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, glaucoma, optic neuropathy, and trauma.
[00118] A
"therapeutically effective amount" used herein refers to the amount of a compound that, when administered to a subject for treatment of a disease or condition, is sufficient to affect such treatment.
Pluripotent Stem Cells A. Embryonic Stem Cells [00119] ES cells are derived from the inner cell mass of blastocysts and have a high in vitro differentiating capability. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo, then culturing the inner mass cells on a feeder layer of non-growing cells. The replated cells can continue to proliferate and produce new colonies of ES cells which can be removed, dissociated, replated again and allowed to grow.
This process of "subculturing" undifferentiated ES cells can be repeated a number of times to produce cell lines containing undifferentiated ES cells (U.S. Patent Nos.
5,843,780; 6,200,806;
7,029,913).
[00120]
Methods for producing mouse ES cells are well known. In one method, a preimplantation blastocyst from the 129 strain of mice is treated with mouse antiserum to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically-inactivated mouse embryonic fibroblasts in medium containing fetal calf serum.
Colonies of undifferentiated ES cells that develop are subcultured on mouse embryonic fibroblast feeder layers in the presence of fetal calf serum to produce populations of ES cells.
In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000).
In other methods, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).
[00121]
Human ES cells can be produced or derived from a zygote or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, pathogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce an embryonic cell by previously described methods (Thomson and Marshall, 1998; Reubinoff et al., 2000). In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are lysed and removed from the inner cell mass which is cultured on a feeder layer of mouse embryonic fibroblasts. Further, clumps of cells derived from the inner cell mass are chemically or mechanically dissociated, replated, and colonies with undifferentiated morphology are selected by micropipette, dissociated, and replated (U.S. Patent No. 6,833,269). In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MATRIGELI'm or laminin in the presence of "conditioned"
medium containing basic fibroblast growth factor (Xu etal., 2001).
[00122] ES
cells can also be derived from other organisms including rhesus monkey and marmoset by previously described methods (Thomson, and Marshall, 1998;
Thomson et al., 1995; Thomson and Odorico, 2000), as well as from established mouse and human cell lines. For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14 and ACT30. As a further example, mouse ES cell lines that have been established include the CGR8 cell line established from the inner cell mass of the mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without feeder layers.
[00123] ES
stem cells can be detected by protein markers including transcription factor 0ct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), 50X2, or REX1.
B. Induced Pluripotent Stem Cells [00124] The induction of pluripotency was originally achieved in 2006 using mouse cells (Yamanaka et al. 2006) and in 2007 using human cells (Yu et al.
2007; Takahashi etal. 2007) by reprogramming of somatic cells via the introduction of transcription factors that are linked to pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into any adult cell type.
[00125]
With the exception of germ cells, any somatic cell can be used as a starting point for iPSCs. For example, cell types could be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or stomach cells. T cells may also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648).
There is no limitation on the degree of cell differentiation or the age of an animal from which cells are collected; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used as sources of somatic cells in the methods disclosed herein. In one embodiment, the somatic cell is itself a PRP cell, such as a human PRP cell. The PRP cell can be an adult or a fetal PRP cell. iPSCs can be grown under conditions that are known to differentiate human ES cells into specific cell types, and express human ES cell markers including: SSEA-1, SSEA-3, S SEA-4, TRA-1-60, and TRA-1-81.
[00126]
Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to one of skill in the art. One of skill in the art can readily produce induced pluripotent stem cells; see for example, Published U.S. Patent Application No. 20090246875, Published U.S. Patent Application No. 2010/0210014; Published U.S.
Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No.
8,129,187;
U.S. Patent No. 8,278,620; PCT Publication NO. WO 2007/069666 Al, and U.S.
Patent No.
8,268,620, which are incorporated herein by reference. Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell. In some embodiments, at least two, at least three, or at least four, of Klf4, c-Myc, 0ct3/4, 5ox2, Nanog, and Lin28 are utilized. In other embodiments, 0ct3/4, 5ox2, c-Myc and Klf4 are utilized.
[00127] The cells are treated with a nuclear reprogramming substance, which is generally one or more factor(s) capable of inducing an iPSC from a somatic cell or a nucleic acid that encodes these substances (including forms integrated in a vector).
The nuclear reprogramming substances generally include at least 0ct3/4, Klf4 and 5ox2 or nucleic acids that encode these molecules. A functional inhibitor of p53, L-myc or a nucleic acid that encodes L-myc, and Lin28 or Lin28b or a nucleic acid that encodes Lin28 or Lin28b, can be utilized as additional nuclear reprogramming substances. Nanog can also be utilized for nuclear reprogramming. As disclosed in published U.S. Patent Application No.
20120196360, exemplary reprogramming factors for the production of iPSCs include (1) 0ct3/4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Sox15, Sox17 or Sox18; Klf4 is replaceable with Klfl, Klf2 or Klf5); (2) 0ct3/4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen (SV4OLT);
(3) 0ct3/4, Klf4, Sox2, L-Myc, TERT, human papilloma virus (HPV)16 E6; (4) 0ct3/4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) 0ct3/4, Klf4, Sox2, L- Myc, TERT, HPV16 E6, E7; (6) 0ct3/4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) 0ct3/4, Klf4, Sox2, L-Myc, Lin28; (8) 0ct3/4, Klf4, 5ox2, L-Myc, Lin28, SV4OLT; (9) 0ct3/4, Klf4, 5ox2, L-Myc, Lin28, TERT, SV4OLT; (10) 0ct3/4, Klf4, 5ox2, L-Myc, SV4OLT; (11) 0ct3/4, Esrrb, 5ox2, L-Myc (Esrrb is replaceable with Esrrg); (12) 0ct3/4, Klf4, 5ox2; (13) 0ct3/4, Klf4, 5ox2, TERT, SV4OLT;
(14) 0ct3/4, Klf4, 5ox2, TERT, HP VI 6 E6; (15) 0ct3/4, Klf4, 5ox2, TERT, HPV16 E7; (16) 0ct3/4, Klf4, 5ox2, TERT, HPV16 E6, HPV16 E7; (17) 0ct3/4, Klf4, 5ox2, TERT, Bmil; (18) 0ct3/4, Klf4, 5ox2, Lin28 (19) 0ct3/4, Klf4, 5ox2, Lin28, SV4OLT; (20) 0ct3/4, Klf4, 5ox2, Lin28, TERT, SV4OLT; (21) 0ct3/4, Klf4, 5ox2, SV4OLT; or (22) 0ct3/4, Esrrb, 5ox2 (Esrrb is replaceable with Esrrg). In one non-limiting example, 0ct3/4, Klf4, 5ox2, and c-Myc are utilized. In other embodiments, 0ct4, Nanog, and 5ox2 are utilized; see for example, U.S.
Patent No. 7,682,828, which is incorporated herein by reference. These factors include, but are not limited to, 0ct3/4, Klf4 and 5ox2. In other examples, the factors include, but are not limited to Oct 3/4, Klf4 and Myc. In some non-limiting examples, 0ct3/4, Klf4, c-Myc, and 5ox2 are utilized. In other non-limiting examples, 0ct3/4, Klf4, 5ox2 and Sal 4 are utilized. Factors like Nanog, Lin28, Klf4, or c-Myc can increase reprogramming efficiency and can be expressed from several different expression vectors. For example, an integrating vector such as the EBV
element-based system can be used (U.S. Patent No. 8,546,140). In a further aspect, reprogramming proteins could be introduced directly into somatic cells by protein transduction.
Reprogramming may further comprise contacting the cells with one or more signaling receptors including glycogen synthase kinase 3 (GSK-3) inhibitor, a mitogen-activated protein kinase kinase (MEK) inhibitor, a transforming growth factor beta (TGF-0) receptor inhibitor or signaling inhibitor, leukemia inhibitory factor (LIF), a p53 inhibitor, an NF-kappa B inhibitor, or a combination thereof Those regulators may include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is anticipated that virtually any iPS cells or cell lines may be used.
[00128]
Mouse and human cDNA sequences of these nuclear reprogramming substances are available with reference to the NCBI accession numbers mentioned in WO
2007/069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming substances, or nucleic acids encoding these reprogramming substances, are known in the art, and disclosed for example, in published U.S. Patent Application No.
2012/0196360 and U.S. Patent No. 8,071,369, which both are incorporated herein by reference.
[00129]
Once derived, iPSCs can be cultured in a medium sufficient to maintain pluripotency. The iPSCs may be used with various media and techniques developed to culture pluripotent stem cells, more specifically, embryonic stem cells, as described in U.S. Patent No.
7,442,548 and U.S. Patent Pub. No. 2003/0211603. In the case of mouse cells, the culture is carried out with the addition of Leukemia Inhibitory Factor (LIF) as a differentiation suppression factor to an ordinary medium. In the case of human cells, it is desirable that basic fibroblast growth factor (bFGF) be added in place of LIF. Other methods for the culture and maintenance of iPSCs, as would be known to one of skill in the art, may be used.
[00130] In certain embodiments, undefined conditions may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or a medium that has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state.
In some embodiments, the cell is cultured in the co-presence of mouse embryonic fibroblasts treated with radiation or an antibiotic to terminate the cell division, as feeder cells. Alternately, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using a defined, feeder-independent culture system, such as a TESRTm medium (Ludwig et al., 2006a; Ludwig etal., 2006b) or E8TM medium (Chen et al., 2011).
[00131] In some embodiments, the iPSC can be modified to express exogenous nucleic acids, such as to include an enhancer operably linked to a promoter and a nucleic acid sequence encoding a first marker. Suitable promoters include, but are not limited to, any promoter expressed in photoreceptor cells, such as a rhodopsin kinase promoter. The construct can also include other elements, such as a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription/translation terminator. Generally, it is advantageous to transfect cells with the construct. Suitable vectors for stable transfection include, but are not limited to retroviral vectors, lentiviral vectors and Sendai virus.
[00132] In some embodiments plasmids that encode a marker are composed of:
(1) a high copy number replication origin, (2) a selectable marker, such as, but not limited to, the neo gene for antibiotic selection with kanamycin, (3) transcription termination sequences, including the tyrosinase enhancer and (4) a multicloning site for incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operably linked to the tyrosinase promoter. There are numerous plasmid vectors that are known in the art for inducing a nucleic acid encoding a protein. These include, but are not limited to, the vectors disclosed in U.S. Patent No. 6,103,470; U.S. Patent No. 7,598,364; U.S. Patent No. 7,989,425;
and U.S. Patent No. 6,416,998, which are incorporated herein by reference.
[00133] A viral gene delivery system can be an RNA-based or DNA-based viral vector. An episomal gene delivery system can be a plasmid, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (5V40)-based episomal vector, a bovine papilloma virus (BPV)-based vector, or a lentiviral vector.
[00134]
Markers include, but are not limited to, fluorescence proteins (for example, green fluorescent protein or red fluorescent protein), enzymes (for example, horse radish peroxidase or alkaline phosphatase or firefly/renilla luciferase or nanoluc), or other proteins. A marker may be a protein (including secreted, cell surface, or internal proteins; either synthesized or taken up by the cell); a nucleic acid (such as an mRNA, or enzymatically active nucleic acid molecule) or a polysaccharide. Included are determinants of any such cell components that are detectable by antibody, lectin, probe or nucleic acid amplification reaction that are specific for the marker of the cell type of interest. The markers can also be identified by a biochemical or enzyme assay or biological response that depends on the function of the gene product. Nucleic acid sequences encoding these markers can be operably linked to the tyrosinase enhancer. In addition, other genes can be included, such as genes that may influence stem cell to PRP differentiation, or photoreceptor function, or physiology, or pathology.
1. MHC Haplotype Matching [00135]
Major Histocompatibility Complex (MHC) is the main cause of immune-rejection of allogeneic organ transplants. There are three major class I MHC
haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA loci are highly polymorphic and are distributed over 4 Mb on chromosome 6.
The ability to haplotype the HLA genes within the region is clinically important since this region is associated with autoimmune and infectious diseases and the compatibility of HLA haplotypes between donor and recipient can influence the clinical outcomes of transplantation. HLAs corresponding to MHC class I present peptides from inside the cell and HLAs corresponding to MHC class II present antigens from outside of the cell to T-lymphocytes.
Incompatibility of MHC haplotypes between the graft and the host triggers an immune response against the graft and leads to its rejection. Thus, a patient can be treated with an immunosuppressant to prevent rejection. HLA-matched stem cell lines may overcome the risk of immune rejection.
[00136]
Because of the importance of HLA in transplantation, the HLA loci are usually typed by serology and the polymerase chain reaction (PCR) for identifying favorable donor-recipient pairs. Serological detection of HLA class I and II antigens can be accomplished using a complement mediated lymphocytotoxicity test with purified T or B
lymphocytes. This procedure is predominantly used for matching HLA-A and -B loci. Molecular-based tissue typing can often be more accurate than serologic testing. Low resolution molecular methods such as SSOP (sequence specific oligonucleotide probes) methods, in which PCR
products are tested against a series of oligonucleotide probes, can be used to identify HLA
antigens, and currently these methods are the most common methods used for Class II-HLA
typing. High resolution techniques such as SSP (sequence specific primer) methods which utilize allele specific primers for PCR amplification can identify specific MHC alleles.
[00137] MHC
compatibility between a donor and a recipient increases significantly if the donor cells are HLA homozygous, i.e. contain identical alleles for each antigen-presenting protein. Most individuals are heterozygous for MHC class I
and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can serve as super donors, and grafts generated from their cells can be transplanted in all individuals that are either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype found in high frequency in a population, these cells may have application in transplantation therapies for a large number of individuals.
[00138]
Accordingly, iPSCs can be produced from somatic cells of the subject to be treated, or another subject with the same or substantially the same HLA
type as that of the patient. In one case, the major HLAs (e.g., the three major loci of HLA-A, HLA-B and HLA-DR) of the donor are identical to the major HLAs of the recipient. In some cases, the somatic cell donor may be a super donor; thus, iPSCs derived from a MHC
homozygous super donor may be used to generate PRP cells. Thus, the iPSCs derived from a super donor may be transplanted in subjects that are either homozygous or heterozygous for that haplotype. For example, the iPSCs can be homozygous at two HLA alleles such as HLA-A and HLA-B. As such, iPSCs produced from super donors can be used in the methods disclosed herein, to produce PRP cells that can potentially "match" a large number of potential recipients.
2. Episomal Vectors [00139] In certain aspects, reprogramming factors are expressed from expression cassettes comprised in one or more exogenous episiomal genetic elements (see U.S. Patent Publication 2010/0003757, incorporated herein by reference). Thus, iPSCs can be essentially free of exogenous genetic elements, such as from retroviral or lentiviral vector elements. These iPSCs are prepared by the use of extra-chromosomally replicating vectors (i.e., episomal vectors), which are vectors capable of replicating episomally to make iPSCs essentially free of exogenous vector or viral elements (see U.S. Patent No. 8,546,140, incorporated herein by reference; Yu et al., 2009). A number of DNA viruses, such as adenoviruses, Simian vacuolating virus 40 (5V40) or bovine papilloma virus (BPV), or budding yeast ARS
(Autonomously Replicating Sequences)-containing plasmids replicate extra-chromosomally or episomally in mammalian cells. These episomal plasmids are intrinsically free from all these disadvantages (Bode et al., 2001) associated with integrating vectors. For example, a lymphotrophic herpes virus-based including or Epstein Barr Virus (EBV) as defined above may replicate extra-chromosomally and help deliver reprogramming genes to somatic cells.
Useful EBV elements are OriP and EBNA-1, or their variants or functional equivalents. An additional advantage of episomal vectors is that the exogenous elements will be lost with time after being introduced into cells, leading to self-sustained iPSCs essentially free of these elements.
[00140]
Other extra-chromosomal vectors include other lymphotrophic herpes virus-based vectors. Lymphotrophic herpes virus is a herpes virus that replicates in a lymphoblast (e.g., a human B lymphoblast) and becomes a plasmid for a part of its natural life-cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpes virus.
Exemplary lymphotrophic herpes viruses include, but are not limited to EBV, Kaposi's sarcoma herpes virus (KSHV); Herpes virus saimiri (HS) and Marek's disease virus (MDV). Also, other sources of episome-based vectors are contemplated, such as yeast ARS, adenovirus, 5V40, or BPV.
C. Somatic Cell Nuclear Transfer [00141]
Pluripotent stem cells can be prepared through the method of somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transfer of a donor nucleus into a spindle-free oocyte. In one method, donor fibroblast nuclei from skin fibroblasts of a rhesus macaque are introduced into the cytoplasm of spindle-free, mature metaphase II
rhesus macaque ooctyes by electrofusion (Byrne et al., 2007). The fused oocytes are activated by exposure to ionomycin, and then incubated until the blastocyst stage. The inner cell masses of selected blastocysts are then cultured to produce embryonic stem cell lines.
The embryonic stem cell lines show normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.
III. Photoreceptor Precursor Cells [00142] In some embodiments, neural retinal progenitor (NRP) cells, photoreceptor precursor (PRP) cells, or optic vesicles (OV) are produced in the methods disclosed herein. The cells in the retina that are directly sensitive to light are the photoreceptor cells. Photoreceptors are photosensitive neurons in the outer part of the retina and can be either rods or cones. In the process of phototransduction, the photoreceptor cells convert incident light energy focused by the lens to electric signals which are then sent via the optic nerve to the brain. Vertebrates have two types of photoreceptor cells including cones and rods. Cones are adapted to detect fine detail, central and color vision and function well in bright light. Rods are responsible for peripheral and dim light vision. Neural signals from the rods and cones undergo processing by other neurons of the retina.
[00143] PRP
cells can express markers such as OTX2, CRX, PRDM1 (BLIMP1), NEUROD1, RCVRN, TUBB3 and L1CAM (CD171). PRP cells express several proteins that can serve as markers for detection by the use of methodologies, such as immunocytochemistry, Western blot analysis, flow cytometry, or enzyme-linked immunoassay (ELISA). For example, one characteristic PRP-marker is RCVRN. PRP cells may not express (at any detectable level) the embryonic stem cells markers OCT-4, NANOG or REX-1.
Specifically, expression of these genes is approximately 100-1000 fold lower in PRP cells than in ES cells or iPSC cells, when assessed by quantitative RT-PCR.
[00144] PRP cell markers may be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot-blot hybridization analysis using sequence-specific primers in standard amplification methods using publicly available sequence data (GENBANKO). Expression of tissue-specific markers as detected at the protein or mRNA level is considered positive if the level is at least or about 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-fold, and more particularly more than 10-, 20-, 30, 40-, 50-fold or higher above that of a control cell, such as an undifferentiated pluripotent stem cell or other unrelated cell type.
[00145]
Dysfunction, injury and loss of photoreceptor cells are factors of many eye diseases and disorders including age-related macular degeneration (AMD), hereditary macular degenerations including Best disease, and retinitis pigmentosa. A
potential treatment for such diseases is the transplantation of PRP or photoreceptor cells into the retina of those in need of such treatment. It is speculated that the replenishment of PRP or photoreceptor cells by their transplantation may delay, halt or reverse degradation, improve retinal function and prevent blindness stemming from such conditions. However, obtaining PRP or photoreceptor cells directly from human donors and embryos is a challenge.
[00146] In some embodiments, methods are provided for producing PRP cells from an essentially single cell suspension of PSCs such as human iPSCs. In some embodiments, the PSCs are cultured to pre-confluency to prevent any cell aggregates. In certain aspects, the PSCs are dissociated by incubation with a cell dissociation solution or enzyme, such as exemplified by Versene, Trypsin, ACCUTASETm or TRYPLETm. PSCs can also be dissociated into an essentially single cell suspension by pipetting.
[00147] In addition, Blebbistatin (e.g., about 2.5 [tM) can be added to the medium to increase PSC survival after dissociation into single cells while the cells are not adhered to a culture vessel. A ROCK inhibitor instead of Blebbistatin may alternatively be used to increase PSC survival after dissociation into single cells.
[00148] To efficiently differentiate PRP cells from the single cell PSCs, an accurate count of the input density can increase PRP differentiation efficiency. Thus, the single cell suspension of PSCs is generally counted before seeding. For example, the single cell suspension of PSCs is counted by a hemocytometer or an automated cell counter, such as VICELLO or TC20. The cells may be diluted to a cell density of about 10,000 to about 500,000 cells/mL, about 50,000 to about 200,000 cells/mL, or about 75,000 to about 150,000 cells/mL.
In a non-limiting example, the single cell suspension of PSCs is diluted to a density of about 100,000 cells/mL in a fully defined cultured medium such as ESSENTIAL 8TM
(E8TM) medium.
[00149]
Once a single cell suspension of PSCs is obtained at a known cell density, the cells are generally seeded in an appropriate culture vessel, such as a tissue culture plate, such as a flask, 6-well, 24-well, or 96-well plate. A culture vessel used for culturing the cell(s) can include, but is particularly not limited to: flask, flask for tissue culture, dish, Petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CELLSTACKO Chambers, culture bag, and roller bottle, as long as it is capable of culturing the stem cells therein. The cells may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In a certain embodiment, the culture vessel may be a bioreactor, which may refer to any device or system ex vivo that supports a biologically active environment such that cells can be propagated.
The bioreactor may have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.
[00150] In certain aspects, the PSCs, such as iPSCs, are plated at a cell density appropriate for efficient differentiation. Generally, the cells are plated at a cell density of about 1,000 to about 75,000 cells/cm2, such as of about 5,000 to about 40,000 cells/cm2. In a 6 well plate, the cells may be seeded at a cell density of about 50,000 to about 400,000 cells per well.
In exemplary methods, the cells are seeded at a cell density of about 100,000, about 150,000, about 200,000, about 250,000, about 300,000 or about 350,000 cells per well, such as about 200,000 cells per well.
[00151] The PSCs, such as iPSCs, are generally cultured on culture plates coated by one or more cellular adhesion proteins to promote cellular adhesion while maintaining cell viability. For example, preferred cellular adhesion proteins include extracellular matrix proteins such as vitronectin, laminin, collagen, and/or fibronectin, which may be used to coat a culturing surface as a means of providing a solid support for pluripotent cell growth. The term "extracellular matrix (ECM)" is recognized in the art. Its components can include, but are not limited to, one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin,
of the cells in the OV cell product are positive for CHX10. In some aspects, the cell markers are PAX6 and Ki67. In some aspects, the method further comprises detecting the absence of TYRP1. In some aspects, the method further comprises detecting the expression of one or more markers selected from the group consisting of MITF, CRALBP, BEST1, OTX2, CRX, BLIMP1, NEUROD1, TUBB3, ONECUT1, and CD171/L1CAM.
[0040] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0042] FIG. 1: Candidate Overview. Schematic of iPSC-derived ocular cell differentiation including neural retinal progenitor cells (NRPs), photoreceptor precursor cells (PRPs) and optic vesicles (0Vs). Candidate 1 (C-1) represents PRP
differentiated under adherent 2-dimensional (2-D) conditions (2-D PRP). Candidate 2 (C-2) represents PRP
differentiated under both 2-D and suspension aggregate 3-dimensional (3-D) conditions (Hybrid PRP). Candidate 3 (C-3) represents OVs differentiated under both 2-D
and 3-D
conditions (Hybrid OV). Candidate 4 (C-4) represents NRPs differentiated under conditions.
[0043] FIG. 2: Schematic of the 2-D PRP differentiation process including media phase duration.
[0044] FIGS. 3A-3C: 2-D PRP MACS Enrichment. Flow cytometry analysis of 2-D
PRP for fIII-tubulin (TUBB3) and Recoverin (RCVRN) expression both (FIG. 3A) pre- and (FIG. 3B) post-MACS enrichment. (FIG. 3C) Pre- and post-MACS flow cytometry analysis summary for 2-D PRP differentiations (n=5).
[0045] FIGS. 4A-4D: 2-D PRP Characterization. (FIG. 4A) Image of day 2 post-thaw 2-D PRP aggregates (10x objective). (FIG. 4B) Immunofluorescence staining of day 1 post-thaw 2-D PRPs for TUBB3 and RCVRN (20x objective). (FIG. 4C) Immunofluorescence staining of day 1 post-thaw 2-D PRPs for CRX and RCVRN (20x objective). (FIG.
4D) Single cell gene expression analysis of day 2 post-thaw 2-D PRP aggregates.
[0046] FIG. 5: Schematic of the Hybrid PRP differentiation process.
[0047] FIG. 6: Input iPSC Density Optimization. Flow cytometry analysis of day Hybrid PRP differentiation for Pax6 and Vsx2 with input densities ranging from 1x104 (10K) to 1x106 (1000K) cells per well of a 6 well plate.
[0048] FIG. 7: RM Medium Optimization. Flow cytometry analysis of day 18 RPCs grown in RM medium with or without the addition of FGF2 and SB431542.
[0049] FIGS. 8A-8B: PM Medium Optimization. (FIG. 8A) Morphology of late-stage plated PRP in PM medium with or without the addition of PD0332991 or Activin A. (FIG. 8B) Flow cytometry analysis of late-stage plated C-2 PRP in PM medium with or without the addition of PD0332991 or Activin A.
[0050] FIG. 9: Hybrid PRP MACS Enrichment. Flow cytometry analysis of Hybrid PRP for TUBB3, NESTIN and RCVRN expression both pre- and post-MACS enrichment.
[0051] FIGS. 10A-10D: Hybrid PRP Characterization. (FIG. 10A) Image of day 2 .. post-thaw Hybrid PRP aggregates (10x objective). (FIG. 10B) Immunofluorescence staining of day 1 post-thaw Hybrid PRP for TUBB3 and RCVRN (20x objective). (FIG. 10C) Immunofluorescence staining of day 1 post-thaw Hybrid PRP for CRX and RCVRN
(20x objective). (FIG. 10D). Single cell gene expression analysis of day 2 post-thaw Hybrid PRP
aggregates.
[0052] FIG. 11: Schematic of the Hybrid OV differentiation process.
[0053] FIGS. 12A-12D: Hybrid OV Morphology and Characterization. (FIG. 12A) Image of day 68 differentiating Hybrid OV. (FIG. 12B) Immunofluorescence staining time course of differentiating Hybrid OV for VSX2 and RCVRN. (FIG 12C) Immunofluorescence staining of late-stage Hybrid OV for Gamma-synuclein (SNCG; retinal ganglion cell marker), RCVRN (PRP and photoreceptor marker), Green-sensitive Opsin (OPN1MW; cone marker), cone-arrestin (ARR3; cone marker) and Rhodopsin (RHO; rod marker). (FIG 12D) Flow cytometry time course analysis of differentiating Hybrid OV for VSX2 and RCVRN.
[0054] FIG. 13: Schematic of the 2-D NRP differentiation process.
[0055] FIGS. 14A-14C: 2-D NRP Characterization. (FIG. 14A) Image of day 2 post-thaw NRP aggregates. (FIG. 14B) Immunofluorescence staining of day 1 post-thaw adherent NRP for PAX6 (left) and VSX2 (right). (FIG. 14C) Flow cytometry analysis of day 2 post-thaw NRP for PAX6, PMEL17 and VSX2.
[0056] FIGS. 15A-15B: Flow analyses of surface antigens and recoverin co-expression corresponding to Table 4 was determined using a quadrant flow plot following dual-labeling with recoverin and the antibody against the respective surface antigen.
Additionally, percent population of cells expressing only recoverin (in FITC, x-axis) or only the surface antigen (in APC, y-axis) was evaluated. All plots were gated against unstained (empty) cells and the corresponding isotype control (REA IgGl, MsIgGl, Ms IgG2a, MsIgG2b, MsIgM).
[0057] FIG. 16: Time course of recoverin expression following enrichment with surface antigens. Recoverin expression is greatest at D65 for CD344-enriched PRP.
Enrichment with CD111 and CD230 also yields high percentage of recoverin expression, compared to pre-enriched (pre-MACS) cells.
[0058] FIG. 17: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with surface antigens CD111, CD230 and CD344 at D55.
[0059] FIG. 18: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with surface antigens CD111, CD230 and CD344 at D65.
[0060] FIG. 19: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with surface antigens CD111, CD230 and CD344 at D75.
[0061] FIG. 20: Post-enrichment expression of on-target PRP and off-target retinal cell evaluation following enrichment with CD133 at D75.
[0062] FIG. 21: Flow analyses of depletion markers.
[0063] FIG. 22: Flow cytometry analysis of PAX6 and CHX10 expression with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
[0064] FIG. 23: Percent expression of PAX6 and CHX10 with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
[0065] FIGS. 24A-24B: (FIG. 24A) Flow cytometry analysis of Ki67 and CHX10 expression with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
*Using conjugated CHX10 shifted the plot (higher) but in this case reduced the expression level; it may be possible that the antibody did not label maximum epitopes due to steric hindrance.
(FIG. 24B) Percent expression of Ki67 and CHX10 with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
[0066] FIGS. 25A-25B: (FIG. 25A) Flow cytometry analysis of TYRP1 and PMEL
expression with or without CKI-7 at Day 15 and Day 25 of PRP differentiation.
(FIG. 25B) Percent expression of TYRP1 and PMEL with or without CKI-7 at Day 15 and Day 25 of PRP
differentiation.
[0067] FIGS. 26A-26B: Comparison of early eye field and RPE markers in the presence (FIG. 26A) and absence (FIG. 26B) of RIM.
[0068] FIGS. 27A-27D: Effects of RIM versus No RIM on early (D15) neural retina differentiation across lines on expression of PAX6 (FIG. 27A), CHX10 (FIG.
27B), TRYP10 (FIG. 27C), and PMEL (FIG. 27D).
[0069] FIGS. 28A-28D: Comparison of mid-stage differentiation (--D30) of PRP
in the presence or absence of RIM on expression of PAX6 (FIG. 28A), CHX10 (FIG. 28B), (FIG. 28C), and PMEL (FIG. 28D).
[0070] FIGS. 29A-29B: Expression of on-target PRP characterization markers at Day 75 in the presence (FIG. 29A) and absence (FIG. 29B) of RIM.
[0071] FIG. 30: Expression of off-target retinal cell markers at D75 differentiation in the presence or absence of RIM.
[0072] FIGS. 31A-31B: Recoverin expression is greater than 90% in PRP cells enriched with SUSD2 compared to CD171. (FIG. 31A) SUSD2-enriched RCVRN-positive population. (FIG. 31B) CD171-enriched RCVRN-positive population.
[0073] FIGS. 32A-32E: Maximum recoverin (FIG. 32A) and SUSD2 (FIG. 32B) expression was observed between differentiation day D55 ¨ D65, with recoverin peaking soon after SUSD2. Recoverin and SUSD2 co-expression (FIG. 32C) mimics the SUSD2 alone expression, suggesting that SUSD2-expressing cells also express recoverin while not all recoverin-positive cells express SUSD2. An overlay of the three graphs clearly demonstrates the parallel expression of the dual recoverin/SUSD2 versus the single SUSD2 stain (FIG. 32D).
SUSD2-labeling with post-fixed recoverin labeling. Independent time course studies of recoverin and SUSD2 co-expression from early stage D55 through later stage D105 (FIG. 32E) supports peak SUSD2 expression around D65 but also shows equally elevated expression at D55.
[0074] FIGS. 33A-33C: Percentage recoverin expression increased further post SUSD2-enrichement at D65, D75 and D85 (FIG. 33A), showing highest expression at D65 which also corresponds to early peak SUSD2 and recoverin expression times.
Although percent recoverin-positive cell population decreases from D65 to D75 to D85 (B), SUSD2-enrichment still enhances percentage recoverin-positive cells compared to CD171-enrichment at D85 (FIG. 33B). However, a limitation of SUSD2-enrichment is low cell output, with <25%
of the initial cell input recovered following enrichment (FIG. 33C). This holds true for cells enriched with either SUSD2 or CD171 at D85, whereby only 3% of total input cells are enriched with SUSD2 while only 18% of total input cells are enriched by CD171.
The flow-through (labeled as SUSD2- and CD171-) appeared to contain the majority of the cells, suggesting a possible reduction in expression of both surface markers at later differentiation stages.
[0075] FIGS. 34A-34E: At D65, the height pre-MACS SUSD2 and pre- and post-MACS recoverin expression, CHX10 expression is at its lowest but significantly increases by D75 and D85 (FIG. 34A). Co-expression of recoverin-CHX10 was <3% at D65, rising and falling at D75 and D85, respectively, suggesting a short-lived bipolar cell population (FIG.
34B). NeuroD1 expression was highest at D65 pre- and post-enrichment compared to D75 and D85 (FIG. 34C) and NeuroD1 co-expression was also greatest at D65 and further enhanced post-SUSD2 enrichment (FIG. 34D). An additional experiment (FIG. 34E) confirmed low CHX10 at D55, and also showed substantial reduction in CHX10 after SUSD2 MACS
enrichment at D75.
I. Description of Illustrative Embodiments [0076] In certain embodiments, the present disclosure provides methods for producing ocular cells, including a photoreceptor precursor (PRP) cell population. PRP
cells can be derived from pluripotent stem cells such as ES cells or iPS cells in a defined 2D cell culture without the need for formation of embryoid bodies or selecting colonies of cells. Alternatively, the PRP cells may be derived in a hybrid adherent 2-D and suspension aggregate 3-D culture.
Briefly, the PSCs may be differentiated to anterior neuroectoderm cells which are cultured in a retinal differentiation media comprising a BMP inhibitor for a short period and then further cultured in a retinal differentiation media without the BMP inhibitor.
Interestingly, the inventors found that the removal of the BMP inhibitor enhances the neural retinal differentiation potential of the anterior neuroectoderm cells. The cells may then be further differentiated into retinal progenitor cells (RPCs) in the presence of nicotinamide and the absence of Activin A to direct the cells toward a photoreceptor lineage instead of other retinal lineages, such as retinal pigment epithelial cells. Finally, the RPCs, may be differentiated in the presence of a TGFP inhibitor, a WNT inhibitor, basic FGF, and a y-secretase inhibitor to produce neural retinal progenitor (NRP) cells which may then be differentiated to PRP cells.
Thus, the present disclosure provides a highly efficient and reproducible method of differentiating PSCs into PRP cells.
[0077] The present disclosure also provides methods for the production of PRP
cells or optic vesicles through the combination of a 2D and 3D aggregate culture. The RPCs may be cultured as aggregates in retinal maturation media free of Activin A for a period of time to produce PRP aggregates which may then be dissociated and cultured as a monolayer.
Alternatively, the RPCs may be cultured in the retinal maturation media free of Activin A for an extended period of time to produce the PRP aggregates and eventually produce optic vesicles.
[0078] Further embodiments of the present disclosure provide a method of purifying the population of PRP cells that are obtained by the above methods. The purification method can comprise positive and/or negative selection. For example, cells which express CD171 may be selected for by cell sorting. Therefore, the purification process yields a PRP-enriched cell population that has a greater percentage of PRP cells than the population obtained after differentiation from the RPCs.
[0079] Thus, the present methods are more time- and cost-efficient, and may enable manufacture of PRP-enriched cell populations for therapeutics from a renewable source, stem cells, at a clinical scale. They may be used to uncover mechanisms, new genes, soluble or membrane-bound factors that are important for the development, differentiation, maintenance, survival and function of photoreceptor cells.
[0080] The PRP cells and photoreceptor cells provided herein may be used in a variety of in vivo and in vitro methods. For example, the PRP cells may be used in vivo to treat conditions of the retina, including but not limited to macular degeneration and retinitis pigmentosa. The PRP cells and photoreceptor cells may also be used in vitro in screening assays to identify putative therapeutic or prophylactic treatment candidates. Further embodiments and advantages of the present disclosure are described below.
I. Definitions [0081] The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, a purified population of cells is greater than about 90%
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, or, most preferably, essentially free of other cell types.
[0082] As used herein, "essentially" or "essentially free," in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0083] As used herein in the specification, "a" or "an" may mean one or more.
As used herein in the claim(s), when used in conjunction with the word "comprising,"
the words "a" or "an" may mean one or more than one.
[0084] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.
[0085] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0086] The term "cell population" is used herein to refer to a group of cells, typically of a common type. The cell population can be derived from a common progenitor or may comprise more than one cell type. An "enriched" cell population refers to a cell population derived from a starting cell population (e.g., an unfractionated, heterogeneous cell population) that contains a greater percentage of a specific cell type than the percentage of that cell type in the starting population. The cell populations may be enriched for one or more cell types and depleted of one or more cell types.
[0087] The term "stem cell" refers herein to a cell that under suitable conditions is capable of differentiating into a diverse range of specialized cell types, while under other suitable conditions is capable of self-renewing and remaining in an essentially undifferentiated pluripotent state. The term "stem cell" also encompasses a pluripotent cell, multipotent cell, precursor cell and progenitor cell. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from genital tissue of a fetus. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state by the expression of certain transcription factors associated with pluripotency; these cells are called "induced pluripotent stem cells" or "iPSCs".
[0088] The term "pluripotent" refers to the property of a cell to differentiate into all other cell types in an organism, with the exception of extraembryonic, or placental, cells.
Pluripotent stem cells are capable of differentiating to cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types) even after prolonged culture. A
pluripotent stem cell is an embryonic stem cell derived from the inner cell mass of a blastocyst.
In other embodiments, the pluripotent stem cell is an induced pluripotent stem cell derived by reprogramming somatic cells.
[0089] The term "differentiation" refers to the process by which an unspecialized cell becomes a more specialized type with changes in structural and/or functional properties. The mature cell typically has altered cellular structure and tissue-specific proteins.
[0090] As used herein, "undifferentiated" refers to cells that display characteristic markers and morphological characteristics of undifferentiated cells that clearly distinguish them from terminally differentiated cells of embryo or adult origin.
[0091] "Embryoid bodies (EBs)" are aggregates of pluripotent stem cells that can undergo differentiation into cells of the endoderm, mesoderm, and ectoderm germ layers. The spheroid structures form when pluripotent stem cells are allowed to aggregate under non-adherent culture conditions and thus form EBs in suspension.
[0092] An "isolated" cell has been substantially separated or purified from others cells in an organism or culture. Isolated cells can be, for example, at least 99%, at least 98% pure, at least 95% pure or at least 90% pure.
[0093] An "embryo" refers to a cellular mass obtained by one or more divisions of a zygote or an activated oocyte with an artificially reprogrammed nucleus.
[0094] An "embryonic stem (ES) cell" is an undifferentiated pluripotent cell which is obtained from an embryo in an early stage, such as the inner cell mass at the blastocyst stage, or produced by artificial means (e.g. nuclear transfer) and can give rise to any differentiated cell type in an embryo or an adult, including germ cells (e.g. sperm and eggs).
[0095] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming a somatic cell by expressing or inducing expression of a combination of factors (herein referred to as reprogramming factors). iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, 0ct4 (sometimes referred to as Oct 3/4), 5ox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram a somatic cell to a pluripotent stem cell.
[0096] An "allele" refers to one of two or more forms of a gene. Diploid organisms such as humans contain two copies of each chromosome, and thus carry one allele on each.
[0097] The term "homozygous" is defined as containing two of the same alleles at a particular locus. The term "heterozygous" refers to as containing two different alleles at a particular locus.
[0098] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. A haplotype can be based upon a set of single-nucleotide polymorphisms (SNPs) on a single chromosome and/or the alleles in the major histocompatibility complex.
[0099] As used herein, the term "haplotype-matched" is defined as the cell (e.g. iPS
cell) and the subject being treated share one or more major histocompatibility locus haplotypes.
The haplotype of the subject can be readily determined using assays well known in the art. The haplotype-matched iPS cell can be autologous or allogeneic. The autologous cells which are grown in tissue culture and differentiated to PRP cells inherently are haplotype-matched to the subject.
[00100]
"Substantially the same HLA type" indicates that the Human Leukocyte Antigen (HLA) type of donor matches with that of a patient to the extent that the transplanted cells, which have been obtained by inducing differentiation of iPSCs derived from the donor's somatic cells, can be engrafted when they are transplanted to the patient.
[00101]
"Super donors" are referred to herein as individuals that are homozygous for certain MHC class I and II genes. These homozygous individuals can serve as super donors and their cells, including tissues and other materials comprising their cells, can be transplanted in individuals that are either homozygous or heterozygous for that haplotype.
The super donor can be homozygous for the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ
locus/loci alleles, respectively.
[00102]
"Feeder-free" or "feeder-independent" is used herein to refer to a culture supplemented with cytokines and growth factors (e.g., TGFP, bFGF, LIF) as a replacement for the feeder cell layer. Thus, "feeder-free" or feeder-independent culture systems and media may be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state.
In some cases, feeder-free cultures utilize an animal-based matrix (e.g.
MATRIGELTm) or are grown on a substrate such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to remain in an essentially undifferentiated state without the need for mouse fibroblast "feeder layers."
[00103]
"Feeder layers" are defined herein as a coating layer of cells such as on the bottom of a culture dish. The feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.
[00104] The term "defined" or "fully-defined," when used in relation to a medium, an extracellular matrix, or a culture condition, refers to a medium, an extracellular matrix, or a culture condition in which the chemical composition and amounts of approximately all the components are known. For example, a defined medium does not contain undefined factors such as in fetal bovine serum, bovine serum albumin or human serum albumin.
Generally, a defined medium comprises a basal media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources) which is supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin.
An example of a fully defined medium is Essential 8TM medium.
[00105] For a medium, extracellular matrix, or culture system used with human cells, the term "Xeno-Free (XF)" refers to a condition in which the materials used are not of non-human animal-origin.
[00106]
"Pre-confluent" refers to a cell culture in which the proportion of the culture surface which is covered by cells is about 60-80%. Usually, pre-confluent refers to a culture in which about 70% of the culture surface is covered by cells.
[00107] The term "retinal progenitor cells", also called "retinal precursor cells"
or "RPCs", encompass cells which are competent for generating all cell types of the retina, including neural retina cells, such as rods, cones, photoreceptor precursor cells, as well as cells which can differentiate into RPE.
[00108] The term "neural retinal progenitors" or "NRPs" refers to cells which are restricted in their differentiation potential to neural retina cell types.
[00109] The terms "photoreceptor precursor cells" and "PRP" cells refer to cells differentiated from embryonic stem cells or induced pluripotent stem cells which can differentiate into photoreceptor cells that expresses the cell marker rhodopsin or any of the three cone opsins, and optionally express the rod or cone cGMP. The photoreceptors may be rod and/or cone photoreceptors.
[00110] The term "optic vesicles" or "OVs" refers to cell aggregates or organoids, including PRP cell aggregates, with a morphology comprising optic vesicle structures.
[00111]
"Retinal pigment epithelium" refers to a layer of pigmented cells between the choroid, a layer filled with blood vessels, and the neural retina.
[00112]
"Retinal Induction Medium (RIM)" refers herein to a growth media that comprises a WNT pathway inhibitor and a BMP pathway inhibitor and can result in the differentiation of PSCs to retinal lineage cells. The RIM also comprises a TGFr3 pathway inhibitor, and may comprise IGF-1 and ascorbic acid.
[00113] The "Retinal Differentiation Medium (RD)" is defined herein as a medium that comprises a WNT inhibitor, a TGFr3 inhibitor and a MEK inhibitor and differentiates anterior neuroectoderm cells. The RDM may (i.e., RD1) or may not (i.e., RD2) comprise a BMP inhibitor, and may comprise IGF-1 and ascorbic acid.
[00114] The "Retinal Maturation Medium (RM)" is defined as a growth medium for the culture of retinal cells comprising Nicotinamide and ascorbic acid.
The RM is preferably free of Activin A. The RM may (i.e., RM1) or may not (i.e., RM2) comprise a y secretase inhibitor, such as DAPT, or a TGFr3 inhibitor, such as SB431542, and may comprise IGF-1 and ascorbic acid.
[00115] The "PRP Maturation Medium (PM)" is referred to herein as a growth medium for the culture of PRP cells comprising Nicotinamide and a y secretase inhibitor, such as DAPT. The PM may (i.e., PM1) or may not (i.e., PM2) contain a CDK
inhibitor, such as PD0332291, a TGF-P pathway activator, such as Activin A, or a mitogen, such as retinoic acid.
[00116] The "Photoreceptor Precursor Induction Medium (FDSC)" refers to a growth medium which comprises a TGFr3 inhibitor, a WNT inhibitor, and a y-secretase inhibitor. The FDSC may comprise basic FGF and ascorbic acid.
[00117] The term "retinal degeneration-related disease" is intended to refer to any disease resulting from innate or postnatal retinal degeneration or abnormalities. Examples of retinal degeneration-related diseases include retinal dysplasia, retinal degeneration, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, glaucoma, optic neuropathy, and trauma.
[00118] A
"therapeutically effective amount" used herein refers to the amount of a compound that, when administered to a subject for treatment of a disease or condition, is sufficient to affect such treatment.
Pluripotent Stem Cells A. Embryonic Stem Cells [00119] ES cells are derived from the inner cell mass of blastocysts and have a high in vitro differentiating capability. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo, then culturing the inner mass cells on a feeder layer of non-growing cells. The replated cells can continue to proliferate and produce new colonies of ES cells which can be removed, dissociated, replated again and allowed to grow.
This process of "subculturing" undifferentiated ES cells can be repeated a number of times to produce cell lines containing undifferentiated ES cells (U.S. Patent Nos.
5,843,780; 6,200,806;
7,029,913).
[00120]
Methods for producing mouse ES cells are well known. In one method, a preimplantation blastocyst from the 129 strain of mice is treated with mouse antiserum to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically-inactivated mouse embryonic fibroblasts in medium containing fetal calf serum.
Colonies of undifferentiated ES cells that develop are subcultured on mouse embryonic fibroblast feeder layers in the presence of fetal calf serum to produce populations of ES cells.
In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000).
In other methods, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).
[00121]
Human ES cells can be produced or derived from a zygote or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, pathogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce an embryonic cell by previously described methods (Thomson and Marshall, 1998; Reubinoff et al., 2000). In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are lysed and removed from the inner cell mass which is cultured on a feeder layer of mouse embryonic fibroblasts. Further, clumps of cells derived from the inner cell mass are chemically or mechanically dissociated, replated, and colonies with undifferentiated morphology are selected by micropipette, dissociated, and replated (U.S. Patent No. 6,833,269). In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MATRIGELI'm or laminin in the presence of "conditioned"
medium containing basic fibroblast growth factor (Xu etal., 2001).
[00122] ES
cells can also be derived from other organisms including rhesus monkey and marmoset by previously described methods (Thomson, and Marshall, 1998;
Thomson et al., 1995; Thomson and Odorico, 2000), as well as from established mouse and human cell lines. For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14 and ACT30. As a further example, mouse ES cell lines that have been established include the CGR8 cell line established from the inner cell mass of the mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without feeder layers.
[00123] ES
stem cells can be detected by protein markers including transcription factor 0ct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), 50X2, or REX1.
B. Induced Pluripotent Stem Cells [00124] The induction of pluripotency was originally achieved in 2006 using mouse cells (Yamanaka et al. 2006) and in 2007 using human cells (Yu et al.
2007; Takahashi etal. 2007) by reprogramming of somatic cells via the introduction of transcription factors that are linked to pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into any adult cell type.
[00125]
With the exception of germ cells, any somatic cell can be used as a starting point for iPSCs. For example, cell types could be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or stomach cells. T cells may also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648).
There is no limitation on the degree of cell differentiation or the age of an animal from which cells are collected; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used as sources of somatic cells in the methods disclosed herein. In one embodiment, the somatic cell is itself a PRP cell, such as a human PRP cell. The PRP cell can be an adult or a fetal PRP cell. iPSCs can be grown under conditions that are known to differentiate human ES cells into specific cell types, and express human ES cell markers including: SSEA-1, SSEA-3, S SEA-4, TRA-1-60, and TRA-1-81.
[00126]
Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to one of skill in the art. One of skill in the art can readily produce induced pluripotent stem cells; see for example, Published U.S. Patent Application No. 20090246875, Published U.S. Patent Application No. 2010/0210014; Published U.S.
Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No.
8,129,187;
U.S. Patent No. 8,278,620; PCT Publication NO. WO 2007/069666 Al, and U.S.
Patent No.
8,268,620, which are incorporated herein by reference. Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell. In some embodiments, at least two, at least three, or at least four, of Klf4, c-Myc, 0ct3/4, 5ox2, Nanog, and Lin28 are utilized. In other embodiments, 0ct3/4, 5ox2, c-Myc and Klf4 are utilized.
[00127] The cells are treated with a nuclear reprogramming substance, which is generally one or more factor(s) capable of inducing an iPSC from a somatic cell or a nucleic acid that encodes these substances (including forms integrated in a vector).
The nuclear reprogramming substances generally include at least 0ct3/4, Klf4 and 5ox2 or nucleic acids that encode these molecules. A functional inhibitor of p53, L-myc or a nucleic acid that encodes L-myc, and Lin28 or Lin28b or a nucleic acid that encodes Lin28 or Lin28b, can be utilized as additional nuclear reprogramming substances. Nanog can also be utilized for nuclear reprogramming. As disclosed in published U.S. Patent Application No.
20120196360, exemplary reprogramming factors for the production of iPSCs include (1) 0ct3/4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Sox15, Sox17 or Sox18; Klf4 is replaceable with Klfl, Klf2 or Klf5); (2) 0ct3/4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen (SV4OLT);
(3) 0ct3/4, Klf4, Sox2, L-Myc, TERT, human papilloma virus (HPV)16 E6; (4) 0ct3/4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) 0ct3/4, Klf4, Sox2, L- Myc, TERT, HPV16 E6, E7; (6) 0ct3/4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) 0ct3/4, Klf4, Sox2, L-Myc, Lin28; (8) 0ct3/4, Klf4, 5ox2, L-Myc, Lin28, SV4OLT; (9) 0ct3/4, Klf4, 5ox2, L-Myc, Lin28, TERT, SV4OLT; (10) 0ct3/4, Klf4, 5ox2, L-Myc, SV4OLT; (11) 0ct3/4, Esrrb, 5ox2, L-Myc (Esrrb is replaceable with Esrrg); (12) 0ct3/4, Klf4, 5ox2; (13) 0ct3/4, Klf4, 5ox2, TERT, SV4OLT;
(14) 0ct3/4, Klf4, 5ox2, TERT, HP VI 6 E6; (15) 0ct3/4, Klf4, 5ox2, TERT, HPV16 E7; (16) 0ct3/4, Klf4, 5ox2, TERT, HPV16 E6, HPV16 E7; (17) 0ct3/4, Klf4, 5ox2, TERT, Bmil; (18) 0ct3/4, Klf4, 5ox2, Lin28 (19) 0ct3/4, Klf4, 5ox2, Lin28, SV4OLT; (20) 0ct3/4, Klf4, 5ox2, Lin28, TERT, SV4OLT; (21) 0ct3/4, Klf4, 5ox2, SV4OLT; or (22) 0ct3/4, Esrrb, 5ox2 (Esrrb is replaceable with Esrrg). In one non-limiting example, 0ct3/4, Klf4, 5ox2, and c-Myc are utilized. In other embodiments, 0ct4, Nanog, and 5ox2 are utilized; see for example, U.S.
Patent No. 7,682,828, which is incorporated herein by reference. These factors include, but are not limited to, 0ct3/4, Klf4 and 5ox2. In other examples, the factors include, but are not limited to Oct 3/4, Klf4 and Myc. In some non-limiting examples, 0ct3/4, Klf4, c-Myc, and 5ox2 are utilized. In other non-limiting examples, 0ct3/4, Klf4, 5ox2 and Sal 4 are utilized. Factors like Nanog, Lin28, Klf4, or c-Myc can increase reprogramming efficiency and can be expressed from several different expression vectors. For example, an integrating vector such as the EBV
element-based system can be used (U.S. Patent No. 8,546,140). In a further aspect, reprogramming proteins could be introduced directly into somatic cells by protein transduction.
Reprogramming may further comprise contacting the cells with one or more signaling receptors including glycogen synthase kinase 3 (GSK-3) inhibitor, a mitogen-activated protein kinase kinase (MEK) inhibitor, a transforming growth factor beta (TGF-0) receptor inhibitor or signaling inhibitor, leukemia inhibitory factor (LIF), a p53 inhibitor, an NF-kappa B inhibitor, or a combination thereof Those regulators may include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is anticipated that virtually any iPS cells or cell lines may be used.
[00128]
Mouse and human cDNA sequences of these nuclear reprogramming substances are available with reference to the NCBI accession numbers mentioned in WO
2007/069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming substances, or nucleic acids encoding these reprogramming substances, are known in the art, and disclosed for example, in published U.S. Patent Application No.
2012/0196360 and U.S. Patent No. 8,071,369, which both are incorporated herein by reference.
[00129]
Once derived, iPSCs can be cultured in a medium sufficient to maintain pluripotency. The iPSCs may be used with various media and techniques developed to culture pluripotent stem cells, more specifically, embryonic stem cells, as described in U.S. Patent No.
7,442,548 and U.S. Patent Pub. No. 2003/0211603. In the case of mouse cells, the culture is carried out with the addition of Leukemia Inhibitory Factor (LIF) as a differentiation suppression factor to an ordinary medium. In the case of human cells, it is desirable that basic fibroblast growth factor (bFGF) be added in place of LIF. Other methods for the culture and maintenance of iPSCs, as would be known to one of skill in the art, may be used.
[00130] In certain embodiments, undefined conditions may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or a medium that has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state.
In some embodiments, the cell is cultured in the co-presence of mouse embryonic fibroblasts treated with radiation or an antibiotic to terminate the cell division, as feeder cells. Alternately, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using a defined, feeder-independent culture system, such as a TESRTm medium (Ludwig et al., 2006a; Ludwig etal., 2006b) or E8TM medium (Chen et al., 2011).
[00131] In some embodiments, the iPSC can be modified to express exogenous nucleic acids, such as to include an enhancer operably linked to a promoter and a nucleic acid sequence encoding a first marker. Suitable promoters include, but are not limited to, any promoter expressed in photoreceptor cells, such as a rhodopsin kinase promoter. The construct can also include other elements, such as a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription/translation terminator. Generally, it is advantageous to transfect cells with the construct. Suitable vectors for stable transfection include, but are not limited to retroviral vectors, lentiviral vectors and Sendai virus.
[00132] In some embodiments plasmids that encode a marker are composed of:
(1) a high copy number replication origin, (2) a selectable marker, such as, but not limited to, the neo gene for antibiotic selection with kanamycin, (3) transcription termination sequences, including the tyrosinase enhancer and (4) a multicloning site for incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operably linked to the tyrosinase promoter. There are numerous plasmid vectors that are known in the art for inducing a nucleic acid encoding a protein. These include, but are not limited to, the vectors disclosed in U.S. Patent No. 6,103,470; U.S. Patent No. 7,598,364; U.S. Patent No. 7,989,425;
and U.S. Patent No. 6,416,998, which are incorporated herein by reference.
[00133] A viral gene delivery system can be an RNA-based or DNA-based viral vector. An episomal gene delivery system can be a plasmid, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (5V40)-based episomal vector, a bovine papilloma virus (BPV)-based vector, or a lentiviral vector.
[00134]
Markers include, but are not limited to, fluorescence proteins (for example, green fluorescent protein or red fluorescent protein), enzymes (for example, horse radish peroxidase or alkaline phosphatase or firefly/renilla luciferase or nanoluc), or other proteins. A marker may be a protein (including secreted, cell surface, or internal proteins; either synthesized or taken up by the cell); a nucleic acid (such as an mRNA, or enzymatically active nucleic acid molecule) or a polysaccharide. Included are determinants of any such cell components that are detectable by antibody, lectin, probe or nucleic acid amplification reaction that are specific for the marker of the cell type of interest. The markers can also be identified by a biochemical or enzyme assay or biological response that depends on the function of the gene product. Nucleic acid sequences encoding these markers can be operably linked to the tyrosinase enhancer. In addition, other genes can be included, such as genes that may influence stem cell to PRP differentiation, or photoreceptor function, or physiology, or pathology.
1. MHC Haplotype Matching [00135]
Major Histocompatibility Complex (MHC) is the main cause of immune-rejection of allogeneic organ transplants. There are three major class I MHC
haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA loci are highly polymorphic and are distributed over 4 Mb on chromosome 6.
The ability to haplotype the HLA genes within the region is clinically important since this region is associated with autoimmune and infectious diseases and the compatibility of HLA haplotypes between donor and recipient can influence the clinical outcomes of transplantation. HLAs corresponding to MHC class I present peptides from inside the cell and HLAs corresponding to MHC class II present antigens from outside of the cell to T-lymphocytes.
Incompatibility of MHC haplotypes between the graft and the host triggers an immune response against the graft and leads to its rejection. Thus, a patient can be treated with an immunosuppressant to prevent rejection. HLA-matched stem cell lines may overcome the risk of immune rejection.
[00136]
Because of the importance of HLA in transplantation, the HLA loci are usually typed by serology and the polymerase chain reaction (PCR) for identifying favorable donor-recipient pairs. Serological detection of HLA class I and II antigens can be accomplished using a complement mediated lymphocytotoxicity test with purified T or B
lymphocytes. This procedure is predominantly used for matching HLA-A and -B loci. Molecular-based tissue typing can often be more accurate than serologic testing. Low resolution molecular methods such as SSOP (sequence specific oligonucleotide probes) methods, in which PCR
products are tested against a series of oligonucleotide probes, can be used to identify HLA
antigens, and currently these methods are the most common methods used for Class II-HLA
typing. High resolution techniques such as SSP (sequence specific primer) methods which utilize allele specific primers for PCR amplification can identify specific MHC alleles.
[00137] MHC
compatibility between a donor and a recipient increases significantly if the donor cells are HLA homozygous, i.e. contain identical alleles for each antigen-presenting protein. Most individuals are heterozygous for MHC class I
and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can serve as super donors, and grafts generated from their cells can be transplanted in all individuals that are either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype found in high frequency in a population, these cells may have application in transplantation therapies for a large number of individuals.
[00138]
Accordingly, iPSCs can be produced from somatic cells of the subject to be treated, or another subject with the same or substantially the same HLA
type as that of the patient. In one case, the major HLAs (e.g., the three major loci of HLA-A, HLA-B and HLA-DR) of the donor are identical to the major HLAs of the recipient. In some cases, the somatic cell donor may be a super donor; thus, iPSCs derived from a MHC
homozygous super donor may be used to generate PRP cells. Thus, the iPSCs derived from a super donor may be transplanted in subjects that are either homozygous or heterozygous for that haplotype. For example, the iPSCs can be homozygous at two HLA alleles such as HLA-A and HLA-B. As such, iPSCs produced from super donors can be used in the methods disclosed herein, to produce PRP cells that can potentially "match" a large number of potential recipients.
2. Episomal Vectors [00139] In certain aspects, reprogramming factors are expressed from expression cassettes comprised in one or more exogenous episiomal genetic elements (see U.S. Patent Publication 2010/0003757, incorporated herein by reference). Thus, iPSCs can be essentially free of exogenous genetic elements, such as from retroviral or lentiviral vector elements. These iPSCs are prepared by the use of extra-chromosomally replicating vectors (i.e., episomal vectors), which are vectors capable of replicating episomally to make iPSCs essentially free of exogenous vector or viral elements (see U.S. Patent No. 8,546,140, incorporated herein by reference; Yu et al., 2009). A number of DNA viruses, such as adenoviruses, Simian vacuolating virus 40 (5V40) or bovine papilloma virus (BPV), or budding yeast ARS
(Autonomously Replicating Sequences)-containing plasmids replicate extra-chromosomally or episomally in mammalian cells. These episomal plasmids are intrinsically free from all these disadvantages (Bode et al., 2001) associated with integrating vectors. For example, a lymphotrophic herpes virus-based including or Epstein Barr Virus (EBV) as defined above may replicate extra-chromosomally and help deliver reprogramming genes to somatic cells.
Useful EBV elements are OriP and EBNA-1, or their variants or functional equivalents. An additional advantage of episomal vectors is that the exogenous elements will be lost with time after being introduced into cells, leading to self-sustained iPSCs essentially free of these elements.
[00140]
Other extra-chromosomal vectors include other lymphotrophic herpes virus-based vectors. Lymphotrophic herpes virus is a herpes virus that replicates in a lymphoblast (e.g., a human B lymphoblast) and becomes a plasmid for a part of its natural life-cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpes virus.
Exemplary lymphotrophic herpes viruses include, but are not limited to EBV, Kaposi's sarcoma herpes virus (KSHV); Herpes virus saimiri (HS) and Marek's disease virus (MDV). Also, other sources of episome-based vectors are contemplated, such as yeast ARS, adenovirus, 5V40, or BPV.
C. Somatic Cell Nuclear Transfer [00141]
Pluripotent stem cells can be prepared through the method of somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transfer of a donor nucleus into a spindle-free oocyte. In one method, donor fibroblast nuclei from skin fibroblasts of a rhesus macaque are introduced into the cytoplasm of spindle-free, mature metaphase II
rhesus macaque ooctyes by electrofusion (Byrne et al., 2007). The fused oocytes are activated by exposure to ionomycin, and then incubated until the blastocyst stage. The inner cell masses of selected blastocysts are then cultured to produce embryonic stem cell lines.
The embryonic stem cell lines show normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.
III. Photoreceptor Precursor Cells [00142] In some embodiments, neural retinal progenitor (NRP) cells, photoreceptor precursor (PRP) cells, or optic vesicles (OV) are produced in the methods disclosed herein. The cells in the retina that are directly sensitive to light are the photoreceptor cells. Photoreceptors are photosensitive neurons in the outer part of the retina and can be either rods or cones. In the process of phototransduction, the photoreceptor cells convert incident light energy focused by the lens to electric signals which are then sent via the optic nerve to the brain. Vertebrates have two types of photoreceptor cells including cones and rods. Cones are adapted to detect fine detail, central and color vision and function well in bright light. Rods are responsible for peripheral and dim light vision. Neural signals from the rods and cones undergo processing by other neurons of the retina.
[00143] PRP
cells can express markers such as OTX2, CRX, PRDM1 (BLIMP1), NEUROD1, RCVRN, TUBB3 and L1CAM (CD171). PRP cells express several proteins that can serve as markers for detection by the use of methodologies, such as immunocytochemistry, Western blot analysis, flow cytometry, or enzyme-linked immunoassay (ELISA). For example, one characteristic PRP-marker is RCVRN. PRP cells may not express (at any detectable level) the embryonic stem cells markers OCT-4, NANOG or REX-1.
Specifically, expression of these genes is approximately 100-1000 fold lower in PRP cells than in ES cells or iPSC cells, when assessed by quantitative RT-PCR.
[00144] PRP cell markers may be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot-blot hybridization analysis using sequence-specific primers in standard amplification methods using publicly available sequence data (GENBANKO). Expression of tissue-specific markers as detected at the protein or mRNA level is considered positive if the level is at least or about 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-fold, and more particularly more than 10-, 20-, 30, 40-, 50-fold or higher above that of a control cell, such as an undifferentiated pluripotent stem cell or other unrelated cell type.
[00145]
Dysfunction, injury and loss of photoreceptor cells are factors of many eye diseases and disorders including age-related macular degeneration (AMD), hereditary macular degenerations including Best disease, and retinitis pigmentosa. A
potential treatment for such diseases is the transplantation of PRP or photoreceptor cells into the retina of those in need of such treatment. It is speculated that the replenishment of PRP or photoreceptor cells by their transplantation may delay, halt or reverse degradation, improve retinal function and prevent blindness stemming from such conditions. However, obtaining PRP or photoreceptor cells directly from human donors and embryos is a challenge.
[00146] In some embodiments, methods are provided for producing PRP cells from an essentially single cell suspension of PSCs such as human iPSCs. In some embodiments, the PSCs are cultured to pre-confluency to prevent any cell aggregates. In certain aspects, the PSCs are dissociated by incubation with a cell dissociation solution or enzyme, such as exemplified by Versene, Trypsin, ACCUTASETm or TRYPLETm. PSCs can also be dissociated into an essentially single cell suspension by pipetting.
[00147] In addition, Blebbistatin (e.g., about 2.5 [tM) can be added to the medium to increase PSC survival after dissociation into single cells while the cells are not adhered to a culture vessel. A ROCK inhibitor instead of Blebbistatin may alternatively be used to increase PSC survival after dissociation into single cells.
[00148] To efficiently differentiate PRP cells from the single cell PSCs, an accurate count of the input density can increase PRP differentiation efficiency. Thus, the single cell suspension of PSCs is generally counted before seeding. For example, the single cell suspension of PSCs is counted by a hemocytometer or an automated cell counter, such as VICELLO or TC20. The cells may be diluted to a cell density of about 10,000 to about 500,000 cells/mL, about 50,000 to about 200,000 cells/mL, or about 75,000 to about 150,000 cells/mL.
In a non-limiting example, the single cell suspension of PSCs is diluted to a density of about 100,000 cells/mL in a fully defined cultured medium such as ESSENTIAL 8TM
(E8TM) medium.
[00149]
Once a single cell suspension of PSCs is obtained at a known cell density, the cells are generally seeded in an appropriate culture vessel, such as a tissue culture plate, such as a flask, 6-well, 24-well, or 96-well plate. A culture vessel used for culturing the cell(s) can include, but is particularly not limited to: flask, flask for tissue culture, dish, Petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CELLSTACKO Chambers, culture bag, and roller bottle, as long as it is capable of culturing the stem cells therein. The cells may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In a certain embodiment, the culture vessel may be a bioreactor, which may refer to any device or system ex vivo that supports a biologically active environment such that cells can be propagated.
The bioreactor may have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.
[00150] In certain aspects, the PSCs, such as iPSCs, are plated at a cell density appropriate for efficient differentiation. Generally, the cells are plated at a cell density of about 1,000 to about 75,000 cells/cm2, such as of about 5,000 to about 40,000 cells/cm2. In a 6 well plate, the cells may be seeded at a cell density of about 50,000 to about 400,000 cells per well.
In exemplary methods, the cells are seeded at a cell density of about 100,000, about 150,000, about 200,000, about 250,000, about 300,000 or about 350,000 cells per well, such as about 200,000 cells per well.
[00151] The PSCs, such as iPSCs, are generally cultured on culture plates coated by one or more cellular adhesion proteins to promote cellular adhesion while maintaining cell viability. For example, preferred cellular adhesion proteins include extracellular matrix proteins such as vitronectin, laminin, collagen, and/or fibronectin, which may be used to coat a culturing surface as a means of providing a solid support for pluripotent cell growth. The term "extracellular matrix (ECM)" is recognized in the art. Its components can include, but are not limited to, one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin,
- 33 -hyaluronectin, undulin, epiligrin, and kalinin. Other ECM components may include synthetic peptides for adhesion (e.g., RGD or IKVAV motifs), synthetic hydrogels (e.g., PEG, PLGA, etc.) or natural hydrogels, such as alginate. In exemplary methods, the PSCs are grown on culture plates coated with vitronectin. In some embodiments, the cellular adhesion proteins are human proteins.
[00152] The extracellular matrix proteins may be of natural origin and purified from human or animal tissues or, alternatively, the ECM proteins may be genetically engineered recombinant proteins or synthetic in nature. The ECM proteins may be a whole protein or in the form of peptide fragments, native or engineered. Examples of ECM protein that may be useful in the matrix for cell culture include laminin, collagen I, collagen IV, fibronectin and vitronectin. In some embodiments, the matrix composition is xeno-free. For example, in the xeno-free matrix to culture human cells, matrix components of human origin may be used, wherein any non-human animal components may be excluded.
[00153] In some aspects, the total protein concentration in the matrix composition may be about 1 ng/mL to about 1 mg/mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 ug/mL to about 300 ug/mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 ug/mL to about 200 ug/mL.
[00154] Cells, such as PRP
cells or PSCs, can be cultured with the nutrients necessary to support the growth of each specific population of cells.
Generally, the cells are cultured in growth media including a carbon source, a nitrogen source and a buffer to maintain pH. The medium can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, pyruvic acid, buffering agents, pH indicators, and inorganic salts. An exemplary growth medium contains a minimal essential media, such as Dulbecco's Modified Eagle's medium (DMEM) or (E8TM) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Minimal Essential Medium Eagle (MEM) Alpha medium, Dulbecco's modified Eagle medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium.
Additionally, the minimal essential media may be supplemented with additives such as horse, calf or fetal bovine serum. Alternatively, the medium can be serum free. In other cases, the growth media may contain "knockout serum replacement," referred to herein as a serum-free formulation
[00152] The extracellular matrix proteins may be of natural origin and purified from human or animal tissues or, alternatively, the ECM proteins may be genetically engineered recombinant proteins or synthetic in nature. The ECM proteins may be a whole protein or in the form of peptide fragments, native or engineered. Examples of ECM protein that may be useful in the matrix for cell culture include laminin, collagen I, collagen IV, fibronectin and vitronectin. In some embodiments, the matrix composition is xeno-free. For example, in the xeno-free matrix to culture human cells, matrix components of human origin may be used, wherein any non-human animal components may be excluded.
[00153] In some aspects, the total protein concentration in the matrix composition may be about 1 ng/mL to about 1 mg/mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 ug/mL to about 300 ug/mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 ug/mL to about 200 ug/mL.
[00154] Cells, such as PRP
cells or PSCs, can be cultured with the nutrients necessary to support the growth of each specific population of cells.
Generally, the cells are cultured in growth media including a carbon source, a nitrogen source and a buffer to maintain pH. The medium can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, pyruvic acid, buffering agents, pH indicators, and inorganic salts. An exemplary growth medium contains a minimal essential media, such as Dulbecco's Modified Eagle's medium (DMEM) or (E8TM) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Minimal Essential Medium Eagle (MEM) Alpha medium, Dulbecco's modified Eagle medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium.
Additionally, the minimal essential media may be supplemented with additives such as horse, calf or fetal bovine serum. Alternatively, the medium can be serum free. In other cases, the growth media may contain "knockout serum replacement," referred to herein as a serum-free formulation
- 34 -optimized to grow and maintain undifferentiated cells, such as stem cell, in culture.
KNOCKOUTTm serum replacement is disclosed, for example, in U.S. Patent Application No.
2002/0076747, which is incorporated herein by reference. Preferably, the PSCs are cultured in a fully-defined and feeder-free media.
[00155] Accordingly, the single cell PSCs are generally cultured in a fully defined culture medium after plating. In certain aspects, about 18-24 hours after seeding, the medium is aspirated and fresh medium, such as E8TM medium, is added to the culture. In certain aspects, the single cell PSCs are cultured in the fully defined culture medium for about 1, 2 or 3 days after plating. Preferably, the single cells PSCs are cultured in the fully defined culture medium for about 2 days before proceeding with the differentiation process.
[00156] In some embodiments, the medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. WO 98/30679, for example. Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include KNOCKOUTTm Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and GLUTAMAXTm (Gibco).
[00157]
Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 30 to 40 C, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39 C but particularly not limited to them. In one embodiment, the cells are cultured at 37 C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.
A. Differentiation Media Retinal Induction Medium [00158]
After the single cell PSCs have adhered to the culture plate, the cells are preferably cultured in Retinal Induction Medium (RIM) to start the differentiation process into retinal lineage cells. The RIM comprises a WNT pathway inhibitor and can result in the
KNOCKOUTTm serum replacement is disclosed, for example, in U.S. Patent Application No.
2002/0076747, which is incorporated herein by reference. Preferably, the PSCs are cultured in a fully-defined and feeder-free media.
[00155] Accordingly, the single cell PSCs are generally cultured in a fully defined culture medium after plating. In certain aspects, about 18-24 hours after seeding, the medium is aspirated and fresh medium, such as E8TM medium, is added to the culture. In certain aspects, the single cell PSCs are cultured in the fully defined culture medium for about 1, 2 or 3 days after plating. Preferably, the single cells PSCs are cultured in the fully defined culture medium for about 2 days before proceeding with the differentiation process.
[00156] In some embodiments, the medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. WO 98/30679, for example. Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include KNOCKOUTTm Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and GLUTAMAXTm (Gibco).
[00157]
Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 30 to 40 C, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39 C but particularly not limited to them. In one embodiment, the cells are cultured at 37 C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.
A. Differentiation Media Retinal Induction Medium [00158]
After the single cell PSCs have adhered to the culture plate, the cells are preferably cultured in Retinal Induction Medium (RIM) to start the differentiation process into retinal lineage cells. The RIM comprises a WNT pathway inhibitor and can result in the
- 35 -differentiation of PSCs to retinal lineage cells. The RIM additionally comprises a TGFr3 pathway inhibitor and a BMP pathway inhibitor. One exemplary RIM medium is shown in Table 1.
[00159] The RIM can include DMEM and F12 at about a 1:1 ratio. In exemplary methods, a WNT pathway inhibitor is included in the RIM, such as CKI-7, a BMP
pathway inhibitor is included, such as LDN193189, and the TGFr3 pathway inhibitor is included, such as SB431542. For example, the RIM comprises about 5 nM to about 50 nM, such as about 10 nM, of LDN193189, about 0.1 uM to about 5 uM, such as about 0.5 uM, of CKI-7, and about 0.5 uM to about 10 uM, such as about 1 uM, of SB431542. Additionally, the RIM
can include knockout serum replacement, such as about 1% to about 5%, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and insulin growth factor 1 (IGF1). Preferably, the IGF1 is animal free IGF1 (AF-IGF1) and is comprised in the RIM from about 0.1 ng/mL to about 10 ng/mL, such as about 1 ng/mL. The media is aspirated each day and replaced with fresh RIM. The cells are generally cultured in the RIM
for about 1 to about 5 days, such as about 1, 2, 3, 4 or 5 days, such as for about 2 days to produce anterior neuroectoderm cells.
Retinal Differentiation Medium [00160] The anterior neuroectoderm cells can then be cultured in Retinal Differentiation Medium (RD) for further differentiation. The RD comprises a WNT pathway inhibitor, a TGFr3 pathway inhibitor and a MEK inhibitor. In one embodiment, the RD
comprises a WNT pathway inhibitor, such as CKI-7, optionally a BMP pathway inhibitor, such as LDN193189, a TGFr3 pathway inhibitor, such as SB431542, and a MEK
inhibitor, such as PD0325901. Generally, the concentrations of the WNT pathway inhibitor, BMP
pathway inhibitor and TGFr3 pathway inhibitor are higher in the RDM as compared to the RIM, such as about 9 to about 11 times higher, such as about 10 times higher. In exemplary methods, the RD
comprises about 50 nM to about 200 nM, such as about 100 nM of LDN193189, about 1 uM
to about 10 uM, such as about 5 uM, of CKI-7, about 1 uM to about 50 uM, such as about 10 uM, of SB431542, and about 0.1 uM to about 10 uM, such as about 1 uM, 2 uM, 3 uM, 4 uM, 5 uM, 6 uM, 7 uM, 8 uM, or 9 uM of PD0325901. Exemplary RD are shown in Table 1.
[00161] In some aspects, the cells may be first differentiated in the presence of a BMP inhibitor, such as LDN1913189, for a period of time before differentiation in the absence of a BMP inhibitor. First, the anterior neuroectoderm cells are cultured in RD1 comprising a
[00159] The RIM can include DMEM and F12 at about a 1:1 ratio. In exemplary methods, a WNT pathway inhibitor is included in the RIM, such as CKI-7, a BMP
pathway inhibitor is included, such as LDN193189, and the TGFr3 pathway inhibitor is included, such as SB431542. For example, the RIM comprises about 5 nM to about 50 nM, such as about 10 nM, of LDN193189, about 0.1 uM to about 5 uM, such as about 0.5 uM, of CKI-7, and about 0.5 uM to about 10 uM, such as about 1 uM, of SB431542. Additionally, the RIM
can include knockout serum replacement, such as about 1% to about 5%, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and insulin growth factor 1 (IGF1). Preferably, the IGF1 is animal free IGF1 (AF-IGF1) and is comprised in the RIM from about 0.1 ng/mL to about 10 ng/mL, such as about 1 ng/mL. The media is aspirated each day and replaced with fresh RIM. The cells are generally cultured in the RIM
for about 1 to about 5 days, such as about 1, 2, 3, 4 or 5 days, such as for about 2 days to produce anterior neuroectoderm cells.
Retinal Differentiation Medium [00160] The anterior neuroectoderm cells can then be cultured in Retinal Differentiation Medium (RD) for further differentiation. The RD comprises a WNT pathway inhibitor, a TGFr3 pathway inhibitor and a MEK inhibitor. In one embodiment, the RD
comprises a WNT pathway inhibitor, such as CKI-7, optionally a BMP pathway inhibitor, such as LDN193189, a TGFr3 pathway inhibitor, such as SB431542, and a MEK
inhibitor, such as PD0325901. Generally, the concentrations of the WNT pathway inhibitor, BMP
pathway inhibitor and TGFr3 pathway inhibitor are higher in the RDM as compared to the RIM, such as about 9 to about 11 times higher, such as about 10 times higher. In exemplary methods, the RD
comprises about 50 nM to about 200 nM, such as about 100 nM of LDN193189, about 1 uM
to about 10 uM, such as about 5 uM, of CKI-7, about 1 uM to about 50 uM, such as about 10 uM, of SB431542, and about 0.1 uM to about 10 uM, such as about 1 uM, 2 uM, 3 uM, 4 uM, 5 uM, 6 uM, 7 uM, 8 uM, or 9 uM of PD0325901. Exemplary RD are shown in Table 1.
[00161] In some aspects, the cells may be first differentiated in the presence of a BMP inhibitor, such as LDN1913189, for a period of time before differentiation in the absence of a BMP inhibitor. First, the anterior neuroectoderm cells are cultured in RD1 comprising a
- 36 -BMP pathway inhibitor, such as LDN193189, for about 1-3 days, such as 2 days.
Next, the cells are cultured in RD2 that does not comprise a BMP pathway inhibitor. The second step may be for about 5-10 days, such as about 7 days, to continue differentiating the anterior neuroectoderm cells. This method was found to increase expression of VSX2 in the subsequently produced PRP cells. VSX2 is the earliest specific marker of neural RPC within the optic vesicle and cup (Rowan, et al., 2004). VSX2 + retinal progenitors can give rise to all cell types of the neural retina: cones, rods, ganglion cells, amacrine cells, bipolar cells, horizontal cells and Muller glia.
[00162]
Generally, the RD comprises DMEM and F12 at about a 1:1 ratio, knockout serum replacement (e.g., about 1% to about 5%, such as about 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid and IGF1 (e.g., about 1 ng/mL to about 50 ng/mL, such as about 10 ng/mL). In particular methods, the cells are given fresh RD each day after aspiration of the media from the previous day.
Generally, the cells are cultured in the RDM for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, such as for about 7 days to differentiate the anterior neuroectoderm cells towards RPCs.
Retinal Maturation Medium [00163]
Next, the anterior neuroectoderm cells can be even further differentiated and expanded by culturing the cells in Retinal Maturation Medium (RM) to produce RPCs. The RM may comprise nicotinamide. The RM can comprise about 1 mM to about 50 mM, such as about 10 mM, of nicotinamide. The RM may further comprise ascorbic acid, such as 50-500 um, particularly about 100-300 um, such as about 200 um. Preferably, the RM is free of or essentially free of Activin A. Exemplary RM media are shown in Table 1. The RM
(e.g., RM2) may further comprise a y-secretase inhibitor, such as DAPT, basic FGF, and/or a TGFr3 pathway inhibitor, such as SB431542.
[00164] The RM can include DMEM and F12 at about a 1:1 ratio, knockout serum replacement at about 1% to about 5%, such as about 1.5%, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. The medium can be changed daily with room temperature RM. The cells are generally cultured in the RM for about 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 days, such as for about 10 days to derive expanded RPCs.
PRP Maturation Medium (PM)
Next, the cells are cultured in RD2 that does not comprise a BMP pathway inhibitor. The second step may be for about 5-10 days, such as about 7 days, to continue differentiating the anterior neuroectoderm cells. This method was found to increase expression of VSX2 in the subsequently produced PRP cells. VSX2 is the earliest specific marker of neural RPC within the optic vesicle and cup (Rowan, et al., 2004). VSX2 + retinal progenitors can give rise to all cell types of the neural retina: cones, rods, ganglion cells, amacrine cells, bipolar cells, horizontal cells and Muller glia.
[00162]
Generally, the RD comprises DMEM and F12 at about a 1:1 ratio, knockout serum replacement (e.g., about 1% to about 5%, such as about 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid and IGF1 (e.g., about 1 ng/mL to about 50 ng/mL, such as about 10 ng/mL). In particular methods, the cells are given fresh RD each day after aspiration of the media from the previous day.
Generally, the cells are cultured in the RDM for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, such as for about 7 days to differentiate the anterior neuroectoderm cells towards RPCs.
Retinal Maturation Medium [00163]
Next, the anterior neuroectoderm cells can be even further differentiated and expanded by culturing the cells in Retinal Maturation Medium (RM) to produce RPCs. The RM may comprise nicotinamide. The RM can comprise about 1 mM to about 50 mM, such as about 10 mM, of nicotinamide. The RM may further comprise ascorbic acid, such as 50-500 um, particularly about 100-300 um, such as about 200 um. Preferably, the RM is free of or essentially free of Activin A. Exemplary RM media are shown in Table 1. The RM
(e.g., RM2) may further comprise a y-secretase inhibitor, such as DAPT, basic FGF, and/or a TGFr3 pathway inhibitor, such as SB431542.
[00164] The RM can include DMEM and F12 at about a 1:1 ratio, knockout serum replacement at about 1% to about 5%, such as about 1.5%, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. The medium can be changed daily with room temperature RM. The cells are generally cultured in the RM for about 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 days, such as for about 10 days to derive expanded RPCs.
PRP Maturation Medium (PM)
- 37 -[00165] The PRPs may be matured in PRP maturation medium (PM). Exemplary PM medium is shown in Table 1. The PM medium comprises ascorbic acid, nicotinamide, and a y-secretase inhibitor, such as DAPT (e.g., about 1 uM to about 10 uM, such as about 5 uM
of DAPT). The PM (e.g., PM2) may also comprise a CDK inhibitor, such as a inhibitor, such as PD0332991 (e.g., about 1 uM to about 50 uM, such as about 10 uM of PD0332991).
[00166] The PM Medium can include DMEM and F12 at about a 1:1 ratio, knockout serum replacement at about 1% to about 5%, such as about 1.5%, MEM
non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid.
The medium can be changed daily with room temperature PM Medium. The cells are generally cultured in the PM medium for about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or days, such as for about 10 days to derive mature PRP cells.
Photoreceptor Precursor Induction Medium (FDSC) [00167] For further differentiation of the RPCs, the cells are preferably cultured 15 in FDSC Medium. Exemplary FDSC Medium is shown in Table 1. The FDSC Medium comprises a WNT pathway inhibitor, a y-secretase inhibitor, and a TGFr3 pathway inhibitor. In one embodiment, the FDSC comprises a WNT pathway inhibitor, such as CKI-7, a TGFr3 pathway inhibitor, such as SB431542, and a y-secretase inhibitor, such as DAPT. In exemplary methods, the FDSC Medium comprises about 1 uM to about 10 uM, such as about 5 uM, of 20 CKI-7, about 1 uM to about 50 uM, such as about 10 uM, of SB431542, and about 1 uM to about 10 uM, such as about 5 uM of DAPT. The FDSC may also comprise basic FGF.
[00168] The FDSC Medium can include DMEM and F12 at about a 1:1 ratio, knockout serum replacement at about 1% to about 5%, such as about 1.5%, MEM
non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid.
In addition, the medium can comprise basic FGF, such as about 5 ng/mL to about 15 ng/mL, such as about 10 ng/mL. The medium can be changed daily with room temperature FDSC
Medium. The cells are generally cultured in the FDSC for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, such as for about 15 days to derive PRP cells.
Table 1: Exemplary Medium Components Essential 8 Medium
of DAPT). The PM (e.g., PM2) may also comprise a CDK inhibitor, such as a inhibitor, such as PD0332991 (e.g., about 1 uM to about 50 uM, such as about 10 uM of PD0332991).
[00166] The PM Medium can include DMEM and F12 at about a 1:1 ratio, knockout serum replacement at about 1% to about 5%, such as about 1.5%, MEM
non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid.
The medium can be changed daily with room temperature PM Medium. The cells are generally cultured in the PM medium for about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or days, such as for about 10 days to derive mature PRP cells.
Photoreceptor Precursor Induction Medium (FDSC) [00167] For further differentiation of the RPCs, the cells are preferably cultured 15 in FDSC Medium. Exemplary FDSC Medium is shown in Table 1. The FDSC Medium comprises a WNT pathway inhibitor, a y-secretase inhibitor, and a TGFr3 pathway inhibitor. In one embodiment, the FDSC comprises a WNT pathway inhibitor, such as CKI-7, a TGFr3 pathway inhibitor, such as SB431542, and a y-secretase inhibitor, such as DAPT. In exemplary methods, the FDSC Medium comprises about 1 uM to about 10 uM, such as about 5 uM, of 20 CKI-7, about 1 uM to about 50 uM, such as about 10 uM, of SB431542, and about 1 uM to about 10 uM, such as about 5 uM of DAPT. The FDSC may also comprise basic FGF.
[00168] The FDSC Medium can include DMEM and F12 at about a 1:1 ratio, knockout serum replacement at about 1% to about 5%, such as about 1.5%, MEM
non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid.
In addition, the medium can comprise basic FGF, such as about 5 ng/mL to about 15 ng/mL, such as about 10 ng/mL. The medium can be changed daily with room temperature FDSC
Medium. The cells are generally cultured in the FDSC for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, such as for about 15 days to derive PRP cells.
Table 1: Exemplary Medium Components Essential 8 Medium
- 38 -Component Vendor Cat# Final Conc.
Essential 8TM Basal Medium Thermo A1517001 98%
Fisher Essential 8TM Supplement Thermo 2%
Fisher Essential 8 Thawing Medium Component Vendor Cat# Final Conc.
Complete Essential 8TM Medium Thermo As prepared 100%
Fisher above Rho Kinase Inhibitor (H1152) Millipore 555550 1 p,M
Sigma Essential 8 Plating Medium Component Vendor Cat# Final Conc.
Complete Essential 8TM Medium Thermo As prepared 100%
Fisher above Blebbistatin Millipore B0560 2.5 p,M
Sigma Retinal Induction Medium (RIM) Component Vendor Cat# Final Conc.
DMEM/F 12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 200p,M
Sigma LDN-193189 Stemgent 04-0074 lOnM
SB 431542 R&D 1614/10 1.0p,M
Systems CKI-7 Dihydrochloride Millipore C0742 0.5p,M
Sigma AF-IGF-1 R&D AFL291 lng/ml Systems Retinal Differentiation Medium #1 (RD1) Component Vendor Cat# Final Conc.
Essential 8TM Basal Medium Thermo A1517001 98%
Fisher Essential 8TM Supplement Thermo 2%
Fisher Essential 8 Thawing Medium Component Vendor Cat# Final Conc.
Complete Essential 8TM Medium Thermo As prepared 100%
Fisher above Rho Kinase Inhibitor (H1152) Millipore 555550 1 p,M
Sigma Essential 8 Plating Medium Component Vendor Cat# Final Conc.
Complete Essential 8TM Medium Thermo As prepared 100%
Fisher above Blebbistatin Millipore B0560 2.5 p,M
Sigma Retinal Induction Medium (RIM) Component Vendor Cat# Final Conc.
DMEM/F 12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 200p,M
Sigma LDN-193189 Stemgent 04-0074 lOnM
SB 431542 R&D 1614/10 1.0p,M
Systems CKI-7 Dihydrochloride Millipore C0742 0.5p,M
Sigma AF-IGF-1 R&D AFL291 lng/ml Systems Retinal Differentiation Medium #1 (RD1) Component Vendor Cat# Final Conc.
- 39 -DMEM/F 12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma LDN-193189 Stemgent 04-0074 100nM
SB 431542 R&D 1614/10 1004 Systems CKI-7 Dihydrochloride Millipore C0742 5p,M
Sigma AF-IGF-1 R&D AFL291 1 Ong/ml Systems PD0325901 Stemgent 04-0006 1 nM
Retinal Differentiation Medium #2 (RD2) Component Vendor Cat# Final Conc.
DMEM/F 12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma SB 431542 R&D 1614/10 1004 Systems CKI-7 Dihydrochloride Millipore C0742 5p,M
Sigma AF-IGF-1 R&D AFL291 1 Ong/ml Systems PD0325901 Stemgent 04-0006 1 nM
Retinal Maturation Medium #1 (RM1)
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma LDN-193189 Stemgent 04-0074 100nM
SB 431542 R&D 1614/10 1004 Systems CKI-7 Dihydrochloride Millipore C0742 5p,M
Sigma AF-IGF-1 R&D AFL291 1 Ong/ml Systems PD0325901 Stemgent 04-0006 1 nM
Retinal Differentiation Medium #2 (RD2) Component Vendor Cat# Final Conc.
DMEM/F 12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma SB 431542 R&D 1614/10 1004 Systems CKI-7 Dihydrochloride Millipore C0742 5p,M
Sigma AF-IGF-1 R&D AFL291 1 Ong/ml Systems PD0325901 Stemgent 04-0006 1 nM
Retinal Maturation Medium #1 (RM1)
- 40 -Component Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma Retinal Maturation Medium #2 (RM2) Component Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma basic FGF R&D AFL233 10-Systems 10Ong/mL
SB 431542 R&D 1614/10 1004 Systems PRP Maturation Medium #1 (PM!) Component Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma Retinal Maturation Medium #2 (RM2) Component Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma basic FGF R&D AFL233 10-Systems 10Ong/mL
SB 431542 R&D 1614/10 1004 Systems PRP Maturation Medium #1 (PM!) Component Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher
- 41 -MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma DAPT Millipore D5942 5p,M
Sigma PRP Maturation Medium #1 (PM2) Component Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma DAPT Millipore D5942 5p,M
Sigma PD0332991 Tocris 4786 10 nIVI
Photoreceptor Precursor Induction Medium (FDSC) Cont Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma DAPT Millipore D5942 5p,M
Sigma PRP Maturation Medium #1 (PM2) Component Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 20004 Sigma Nicotinamide Millipore N0636 10mM
Sigma DAPT Millipore D5942 5p,M
Sigma PD0332991 Tocris 4786 10 nIVI
Photoreceptor Precursor Induction Medium (FDSC) Cont Vendor Cat# Final Conc.
DMEM/F12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher
- 42 -CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 200p,M
Sigma basic FGF R&D AFL233 lOng/mL
Systems DAPT Millipore D5942 5p,M
Sigma SB 431542 R&D 1614/10 10p,M
Systems CKI-7 Dihydrochloride Millipore C0742 5p,M
Sigma MACS Buffer Component Vendor Cat# Final Conc.
DPBS (without calcium and Thermo 14190-144 98%
magnesium) Fisher Fetal Bovine Serum GE Life 5H30071.03 2%
Sciences UltraPureTM EDTA Solution Thermo 15575-020 2mM
Fisher Post-thaw Medium #1 (PT!) Component Vendor Cat# Final Conc.
Neurobasal CTS Grade Thermo A13712-01 99%
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher Glutamax Thermo 35050-061 1%
Fisher Y-27632 Tocris 1254/10 10 p,M
Post-thaw Medium #2 (PT2) Component Vendor Cat# Final Conc.
DMEM/F 12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher Ascorbic Acid Millipore A4544 200p,M
Sigma basic FGF R&D AFL233 lOng/mL
Systems DAPT Millipore D5942 5p,M
Sigma SB 431542 R&D 1614/10 10p,M
Systems CKI-7 Dihydrochloride Millipore C0742 5p,M
Sigma MACS Buffer Component Vendor Cat# Final Conc.
DPBS (without calcium and Thermo 14190-144 98%
magnesium) Fisher Fetal Bovine Serum GE Life 5H30071.03 2%
Sciences UltraPureTM EDTA Solution Thermo 15575-020 2mM
Fisher Post-thaw Medium #1 (PT!) Component Vendor Cat# Final Conc.
Neurobasal CTS Grade Thermo A13712-01 99%
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher Glutamax Thermo 35050-061 1%
Fisher Y-27632 Tocris 1254/10 10 p,M
Post-thaw Medium #2 (PT2) Component Vendor Cat# Final Conc.
DMEM/F 12 Thermo 11330-032 99%
Fisher CTSTm KnockOutTM SR Thermo A1099201 1.50%
XenoFree Kit Fisher MEM non-essential AA Thermo 11140 0.1mM
Fisher Sodium Pyruvate Thermo 11360-070 1mM
Fisher CTSTm N-2 Supplement Thermo A13707-01 1%
Fisher B-27 Supplement (+VitA) Thermo 17504-044 2%
Fisher
- 43 -Ascorbic Acid Millipore A4544 200p,M
Sigma Nicotinamide Millipore N0636 10mM
Sigma Y-27632 (optional) Tocris 1254/10 10 p,M
[00169] In addition, Blebbistatin (e.g., about 2.5 p.M) can be added to the medium to increase PRP survival and maintain purity by promoting aggregate formation. A
ROCK inhibitor instead of Blebbistatin may alternatively be used to increase PRP survival after dissociation into single cells, such as by using TRYPLETm.
[00170] The PRP aggregates may be cultured to produce hybrid photoreceptor cells or optic vesicles.
B. Cryopreservation of PRP Cells [00171] The photoreceptor precursor cells produced by the methods disclosed herein can be cryopreserved, see for example, PCT Publication No. 2012/149484 A2, which is incorporated by reference herein. The cells can be cryopreserved with or without a substrate.
In several embodiments, the storage temperature ranges from about -50 C to about -60 C, about -60 C to about -70 C, about -70 C to about -80 C, about -80 C to about -90 C, about -90 C to about - 100 C, and overlapping ranges thereof In some embodiments, lower temperatures are used for the storage (e.g., maintenance) of the cryopreserved cells. In several embodiments, liquid nitrogen (or other similar liquid coolant) is used to store the cells. In further embodiments, the cells are stored for greater than about 6 hours. In additional embodiments, the cells are stored about 72 hours. In several embodiments, the cells are stored 48 hours to about one week. In yet other embodiments, the cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, the cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. The cells can also be stored for longer times. The cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.
[00172] In some embodiments, additional cryoprotectants can be used. For example, the cells can be cryopreserved in a cryopreservation solution comprising one or more cryoprotectants, such as DM80, serum albumin, such as human or bovine serum albumin. In certain embodiments, the solution comprises about 1 %, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%=, about 8%, about 9%, or about 10%
DMSO. In other embodiments, the solution comprises about 1% to about 3%, about 2% to
Sigma Nicotinamide Millipore N0636 10mM
Sigma Y-27632 (optional) Tocris 1254/10 10 p,M
[00169] In addition, Blebbistatin (e.g., about 2.5 p.M) can be added to the medium to increase PRP survival and maintain purity by promoting aggregate formation. A
ROCK inhibitor instead of Blebbistatin may alternatively be used to increase PRP survival after dissociation into single cells, such as by using TRYPLETm.
[00170] The PRP aggregates may be cultured to produce hybrid photoreceptor cells or optic vesicles.
B. Cryopreservation of PRP Cells [00171] The photoreceptor precursor cells produced by the methods disclosed herein can be cryopreserved, see for example, PCT Publication No. 2012/149484 A2, which is incorporated by reference herein. The cells can be cryopreserved with or without a substrate.
In several embodiments, the storage temperature ranges from about -50 C to about -60 C, about -60 C to about -70 C, about -70 C to about -80 C, about -80 C to about -90 C, about -90 C to about - 100 C, and overlapping ranges thereof In some embodiments, lower temperatures are used for the storage (e.g., maintenance) of the cryopreserved cells. In several embodiments, liquid nitrogen (or other similar liquid coolant) is used to store the cells. In further embodiments, the cells are stored for greater than about 6 hours. In additional embodiments, the cells are stored about 72 hours. In several embodiments, the cells are stored 48 hours to about one week. In yet other embodiments, the cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, the cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. The cells can also be stored for longer times. The cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.
[00172] In some embodiments, additional cryoprotectants can be used. For example, the cells can be cryopreserved in a cryopreservation solution comprising one or more cryoprotectants, such as DM80, serum albumin, such as human or bovine serum albumin. In certain embodiments, the solution comprises about 1 %, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%=, about 8%, about 9%, or about 10%
DMSO. In other embodiments, the solution comprises about 1% to about 3%, about 2% to
- 44 -about 40o, about 30o to about 5%, about 40o to about 60o, about 5% to about 70o, about 60o to about 8%, about 7% to about 9%, or about 8% to about 1000 dimethylsulfoxide (DMSO) or albumin. In a specific embodiment, the solution comprises 2.50o DMSO. In another specific embodiment, the solution comprises 1000 DMSO.
[00173] Cells may be cooled, for example, at about 1 C/minute during cryopreservation. In some embodiments, the cryopreservation temperature is about -80 C to about -180 C, or about -125 C to about -140 C. In some embodiments, the cells are cooled to 4 C prior to cooling at about 1 C/minute. Cryopreserved cells can be transferred to vapor phase of liquid nitrogen prior to thawing for use. In some embodiments, for example, once the cells have reached about -80 C, they are transferred to a liquid nitrogen storage area.
Cryopreservation can also be done using a controlled-rate freezer.
Cryopreserved cells may be thawed, e.g., at a temperature of about 25 C to about 40 C, and typically at a temperature of about 37 C.
[00174]
Alternatively, the cells may be cryopreserved as aggregates without dissociation into a single cell suspension. For example, following PRP
enrichment, single cells may be allowed to re-aggregate in tissue culture flasks for two days in minimal medium (RMN). Aggregates may be pooled and a sample aliquot obtained for cell counts.
Following a series of washes, aggregates can be resuspended in CryoSTOR CS10 Freeze Medium and the aggregate suspensions may be transferred to liquid nitrogen storage vials, such as at 25x10^6 aggregated cell products/vial.
C. Inhibitors WNT Pathway Inhibitors [00175] WNT
is a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions and are related to the Drosophila segment polarity gene, wingless. In humans, the WNT family of genes encodes 38 to 43 kDa cysteine rich glycoproteins. The WNT proteins have a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence (see e.g., Shimizu eta! Cell Growth Differ 8: 1349-1358 (1997)) and 22 conserved cysteine residues. Because of their ability to promote stabilization of cytoplasmic beta-catenin, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of particular WNT proteins has been shown to be associated with certain cancers.
[00173] Cells may be cooled, for example, at about 1 C/minute during cryopreservation. In some embodiments, the cryopreservation temperature is about -80 C to about -180 C, or about -125 C to about -140 C. In some embodiments, the cells are cooled to 4 C prior to cooling at about 1 C/minute. Cryopreserved cells can be transferred to vapor phase of liquid nitrogen prior to thawing for use. In some embodiments, for example, once the cells have reached about -80 C, they are transferred to a liquid nitrogen storage area.
Cryopreservation can also be done using a controlled-rate freezer.
Cryopreserved cells may be thawed, e.g., at a temperature of about 25 C to about 40 C, and typically at a temperature of about 37 C.
[00174]
Alternatively, the cells may be cryopreserved as aggregates without dissociation into a single cell suspension. For example, following PRP
enrichment, single cells may be allowed to re-aggregate in tissue culture flasks for two days in minimal medium (RMN). Aggregates may be pooled and a sample aliquot obtained for cell counts.
Following a series of washes, aggregates can be resuspended in CryoSTOR CS10 Freeze Medium and the aggregate suspensions may be transferred to liquid nitrogen storage vials, such as at 25x10^6 aggregated cell products/vial.
C. Inhibitors WNT Pathway Inhibitors [00175] WNT
is a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions and are related to the Drosophila segment polarity gene, wingless. In humans, the WNT family of genes encodes 38 to 43 kDa cysteine rich glycoproteins. The WNT proteins have a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence (see e.g., Shimizu eta! Cell Growth Differ 8: 1349-1358 (1997)) and 22 conserved cysteine residues. Because of their ability to promote stabilization of cytoplasmic beta-catenin, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of particular WNT proteins has been shown to be associated with certain cancers.
- 45 -[00176] A
WNT inhibitor (also referred to as a WNT pathway inhibitor) herein refers to WNT inhibitors in general. Thus, a WNT inhibitor refers to any inhibitor of a member of the WNT family proteins including Wntl, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wntl0a, Wntl 1, and Wnt16. Certain embodiments of the present methods concern a WNT inhibitor in the differentiation medium.
Examples of suitable WNT inhibitors, already known in the art, include N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulphonamide dihydrochloride (CKI-7), N-(6-Methy1-2-benzothiazoly1)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno [3,2-d] pyrimidin-2-yl)thio] -acetami de (IWP2), N-(6-Methyl-2 -b enzothi azoly1)-2- [(3,4,6,7-tetrahy dro-3 -(2 -methoxy pheny1)-4 -oxothi eno [3,2-d] py ri mi din-2 -yl)thi ol -acetami de (IWP4), 2-Phenoxybenzoic acid-R5-methyl-2-furanyOmethylenelhydrazide (PNU 74654) 2,4-diamino-quinazoline, quercetin, 3,5,7,8-Tetrahy dro-244-(trifluoromethy Ophenyll -4H-thi opy rano [4,3-d] pyrimi din-4 -one (XAV 939), 2,5-Dichloro-N-(2-methy1-4-nitrophenyl)benzenesulfonamide (FH 535), N-[442-Ethy1-4-(3-methylpheny1)-5-thiazoly1]-2-pyridinyllbenzamide (TAK 715), Dickkopf-related protein one (DKK1), and Secreted frizzled-related protein (SFRP1) 1. In addition, inhibitors of WNT can include antibodies to, dominant negative variants of, and siRNA and antisense nucleic acids that suppress expression of WNT. Inhibition of WNT can also be achieved using RNA-mediated interference (RNAi).
BMP Pathway Inhibitors [00177] Bone morphogenic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGF13) superfamily. BMPs are considered to constitute a group of pivotal morphogenetic signals, orchestrating architecture through the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes.
[00178] BMP pathway inhibitors (also referred to herein as BMP inhibitors) may include inhibitors of BMP signaling in general or inhibitors specific for BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10 or BMP15. Exemplary BMP
inhibitors include 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-alpyrimidin-3-yOquinoline hydrochloride (LDN193189), 6-[4- [2 -(1 -Pi peridinyl)ethoxy] phenyl] -3 -(4-py ri diny1)-pyrazolo [1,5-al py rimidine dihy drochl ori de (Dorsomorphin), 4-[6-[4-(1-Methylethoxy)phenyllpyrazolo [1,5 -a] pyrimi din-3 -yl] -quinoline (DMH1), 4 - [6- [4- [2-(4-
WNT inhibitor (also referred to as a WNT pathway inhibitor) herein refers to WNT inhibitors in general. Thus, a WNT inhibitor refers to any inhibitor of a member of the WNT family proteins including Wntl, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wntl0a, Wntl 1, and Wnt16. Certain embodiments of the present methods concern a WNT inhibitor in the differentiation medium.
Examples of suitable WNT inhibitors, already known in the art, include N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulphonamide dihydrochloride (CKI-7), N-(6-Methy1-2-benzothiazoly1)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno [3,2-d] pyrimidin-2-yl)thio] -acetami de (IWP2), N-(6-Methyl-2 -b enzothi azoly1)-2- [(3,4,6,7-tetrahy dro-3 -(2 -methoxy pheny1)-4 -oxothi eno [3,2-d] py ri mi din-2 -yl)thi ol -acetami de (IWP4), 2-Phenoxybenzoic acid-R5-methyl-2-furanyOmethylenelhydrazide (PNU 74654) 2,4-diamino-quinazoline, quercetin, 3,5,7,8-Tetrahy dro-244-(trifluoromethy Ophenyll -4H-thi opy rano [4,3-d] pyrimi din-4 -one (XAV 939), 2,5-Dichloro-N-(2-methy1-4-nitrophenyl)benzenesulfonamide (FH 535), N-[442-Ethy1-4-(3-methylpheny1)-5-thiazoly1]-2-pyridinyllbenzamide (TAK 715), Dickkopf-related protein one (DKK1), and Secreted frizzled-related protein (SFRP1) 1. In addition, inhibitors of WNT can include antibodies to, dominant negative variants of, and siRNA and antisense nucleic acids that suppress expression of WNT. Inhibition of WNT can also be achieved using RNA-mediated interference (RNAi).
BMP Pathway Inhibitors [00177] Bone morphogenic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGF13) superfamily. BMPs are considered to constitute a group of pivotal morphogenetic signals, orchestrating architecture through the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes.
[00178] BMP pathway inhibitors (also referred to herein as BMP inhibitors) may include inhibitors of BMP signaling in general or inhibitors specific for BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10 or BMP15. Exemplary BMP
inhibitors include 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-alpyrimidin-3-yOquinoline hydrochloride (LDN193189), 6-[4- [2 -(1 -Pi peridinyl)ethoxy] phenyl] -3 -(4-py ri diny1)-pyrazolo [1,5-al py rimidine dihy drochl ori de (Dorsomorphin), 4-[6-[4-(1-Methylethoxy)phenyllpyrazolo [1,5 -a] pyrimi din-3 -yl] -quinoline (DMH1), 4 - [6- [4- [2-(4-
- 46 -Morpholinyl)ethoxy]phenyl]pyrazolo [1,5-a1pyrimidin-3-yl]quinoline (DMH-2), and 5- [6-(4-Methoxyphenyl)pyrazolo [1,5-a1pyrimidin-3-y11quinoline (ML 347).
TGEfl Pathway Inhibitors [00179]
Transforming growth factor beta (TGF43) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. It is a type of cytokine which plays a role in immunity, cancer, bronchial asthma, lung fibrosis, heart disease, diabetes, and multiple sclerosis. TGF-r3 exists in at least three isoforms called TGF-01, TGF-132 and TGF-03. The TGF-13 family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibins, activin, anti-mtillerian hormone, bone morphogenetic protein, decapentaplegic and Vg-1.
[00180]
TGF13 pathway inhibitors (also referred to herein as TGF13 inhibitors) may include any inhibitors of TGF13 signaling in general. For example, the TGF13 inhibitor is 444-(1,3-benzodioxo1-5-y1)-5-(2-pyridiny1)-1H-imidazol-2-yl]benzamide (SB431542), 642-(1,1-Dimethylethyl)-5-(6-methy1-2-pyridiny1)-1H-imidazol-4-y11quinoxaline (SB525334), 2-(5- Benzo [1,3] dioxo1-5-y1-2-ieri-butyl-3H-imidazol-4-y1)-6-methylpyridine hydrochloride hydrate (SB- 505124), 4-(5-Benzol[1,31dioxol- 5-y1-4-pyridin-2-y1-1H-imidazol-2-y1)-benzamide hydrate, 444-(1,3-Benzodioxo1-5-y1)-5-(2- pyridiny1)-1H-imidazol-2-y11-benzamide hydrate, left-right determination factor (Lefty), 3-(6-Methy1-2-pyridiny1)-N-pheny1-4-(4-quinoliny1)-1H-py razol e-1 -carbothi o ami de (A 83-01), 4-[4-(2,3 -Dihy dro-1,4-benzodioxin-6-y1)-5-(2-pyridiny1)-1H-imidazol-2-y1]benzamide (D 4476), 4-[4-[3-(2-Pyridiny1)-1H-pyrazol-4-y11-2-pyridiny11-N-(tetrahydro-2H-pyran-4-y1)-benzamide (GW
788388), 443-(2-Pyridiny1)-1H-pyrazol-4-y11-quinoline (LY 364847), 442-Fluoro-543-(6-methy1-2-pyridiny1)-1H-pyrazol-4-y11pheny11-1H-pyrazole-1-ethanol (R 268712) or 2-(3-(6-Methy 1pyri dine-2-y1)-1H-py razol-4-y1)-1,5 -naphthy ri dine (Rep S ox).
MEK Inhibitors [00181] A
MEK inhibitor is a chemical or drug that inhibits the mitogen-activated protein kinase enzymes MEK1 or MEK2. They can be used to affect the MAPK/ERK
pathway. For example, MEK inhibitors include N-[(2R)-2,3-Dihydroxypropoxy1-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino1- benzamide (PD0325901), N-[3-[3-cyclopropy1-5-(2-fluoro-4-iodoanilino)-6,8-dimethy1-2,4,7-trioxopyrido [4,3-d] pyrimidin-1 -yl] phenyl] acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-
TGEfl Pathway Inhibitors [00179]
Transforming growth factor beta (TGF43) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. It is a type of cytokine which plays a role in immunity, cancer, bronchial asthma, lung fibrosis, heart disease, diabetes, and multiple sclerosis. TGF-r3 exists in at least three isoforms called TGF-01, TGF-132 and TGF-03. The TGF-13 family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibins, activin, anti-mtillerian hormone, bone morphogenetic protein, decapentaplegic and Vg-1.
[00180]
TGF13 pathway inhibitors (also referred to herein as TGF13 inhibitors) may include any inhibitors of TGF13 signaling in general. For example, the TGF13 inhibitor is 444-(1,3-benzodioxo1-5-y1)-5-(2-pyridiny1)-1H-imidazol-2-yl]benzamide (SB431542), 642-(1,1-Dimethylethyl)-5-(6-methy1-2-pyridiny1)-1H-imidazol-4-y11quinoxaline (SB525334), 2-(5- Benzo [1,3] dioxo1-5-y1-2-ieri-butyl-3H-imidazol-4-y1)-6-methylpyridine hydrochloride hydrate (SB- 505124), 4-(5-Benzol[1,31dioxol- 5-y1-4-pyridin-2-y1-1H-imidazol-2-y1)-benzamide hydrate, 444-(1,3-Benzodioxo1-5-y1)-5-(2- pyridiny1)-1H-imidazol-2-y11-benzamide hydrate, left-right determination factor (Lefty), 3-(6-Methy1-2-pyridiny1)-N-pheny1-4-(4-quinoliny1)-1H-py razol e-1 -carbothi o ami de (A 83-01), 4-[4-(2,3 -Dihy dro-1,4-benzodioxin-6-y1)-5-(2-pyridiny1)-1H-imidazol-2-y1]benzamide (D 4476), 4-[4-[3-(2-Pyridiny1)-1H-pyrazol-4-y11-2-pyridiny11-N-(tetrahydro-2H-pyran-4-y1)-benzamide (GW
788388), 443-(2-Pyridiny1)-1H-pyrazol-4-y11-quinoline (LY 364847), 442-Fluoro-543-(6-methy1-2-pyridiny1)-1H-pyrazol-4-y11pheny11-1H-pyrazole-1-ethanol (R 268712) or 2-(3-(6-Methy 1pyri dine-2-y1)-1H-py razol-4-y1)-1,5 -naphthy ri dine (Rep S ox).
MEK Inhibitors [00181] A
MEK inhibitor is a chemical or drug that inhibits the mitogen-activated protein kinase enzymes MEK1 or MEK2. They can be used to affect the MAPK/ERK
pathway. For example, MEK inhibitors include N-[(2R)-2,3-Dihydroxypropoxy1-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino1- benzamide (PD0325901), N-[3-[3-cyclopropy1-5-(2-fluoro-4-iodoanilino)-6,8-dimethy1-2,4,7-trioxopyrido [4,3-d] pyrimidin-1 -yl] phenyl] acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-
- 47 -hydroxy ethoxy)-3 -methy lb enzi mi dazol e-5 -carb oxami de (MEK162), N- [3 ,4-difluoro-2-(2-fluoro-4-i odoanilino)-6-methoxy phenyl] -1 -(2,3 -dihy droxypropyl)cy cl opropane-1 -sulfonamide (RDEA119), and 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).
Gamma-secretase inhibitors [00182]
Gamma secretase is a multi-subunit protease complex, itself an integral membrane protein, that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. Proteases of this type are known as intramembrane proteases. The most well-known substrate of gamma secretase is amyloid precursor protein, a large integral membrane protein that, when cleaved by both gamma and beta secretase, produces a short amino acid peptide called amyloid beta whose abnormally folded fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.
[00183]
Gamma secretase inhibitors herein refer to y-secretase inhibitors in general. For example, y-secretase inhibitors include, but are not limited to N-[(3,5-Difluorophenyl)acetyl] -L-alany1-2-phenyl]gly cine-1,1-dimethylethyl ester (DAP T), S -Chl oro-N-[(1S)-3 ,3 ,3 -trifluoro-1-(hy droxy methy 0-2-(trifl uoromethy Opropyl] -2-thiophenesulfonamide (Begacestat), MDL-28170,3,5-Bis(4-nitrophenoxy)benzoic acid (Compound W), 7-Amino-4-chloro-3-methoxy-1H-2-benzopyran (JLK6), (5S)-(tert-Butoxy carbonylamino)-6-phenyl-(4R)-hy droxy -(2R)-b enzy lhexanoy1)-L -leucy-L -phenylalaninamide (L-685,485), (R)-2-Fluoro-a-methyl[1,11-bipheny1]-4-acetic acid ((R)-Flurbiprofen; Flurizan), N-[(1S)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5H-dibenz[b,dIazepin-7-yl] amino] -1-methy1-2-oxoethyl] -3,5 -difluorobenzeneacetami de (Dibenzazepine; DBZ), N-[cis-4- [(4-ChlorophenyOsulfony11-4-(2,5-difluorophenyl)cy clohexyl] -1,1,1-trifluoromethanesulfonami de (MRK560), (2S)-2-[[(2S)-6,8-Difluoro-1,2,3,4-tetrahy dro-2-naphtha' enyl] amino] -N41 - [2- [(2,2-dimethylpropyl)amino] -1,1 -di methylethyl] -1H-imi dazol-4-yl]pentanamide dihydrobromide (PF3084014 hydrobromide) and 2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl] (2,5-difluorophenyl)amino] ethyl-5-fluorobenzenebutanoic acid (BMS299897).
Cyclin Dependent Kinase Inhibitors [00184] Cyclin dependent kinases (CDKs) are a family of sugar kinases first discovered for their role in regulating the cell cycle. They are also involved in regulating
Gamma-secretase inhibitors [00182]
Gamma secretase is a multi-subunit protease complex, itself an integral membrane protein, that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. Proteases of this type are known as intramembrane proteases. The most well-known substrate of gamma secretase is amyloid precursor protein, a large integral membrane protein that, when cleaved by both gamma and beta secretase, produces a short amino acid peptide called amyloid beta whose abnormally folded fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.
[00183]
Gamma secretase inhibitors herein refer to y-secretase inhibitors in general. For example, y-secretase inhibitors include, but are not limited to N-[(3,5-Difluorophenyl)acetyl] -L-alany1-2-phenyl]gly cine-1,1-dimethylethyl ester (DAP T), S -Chl oro-N-[(1S)-3 ,3 ,3 -trifluoro-1-(hy droxy methy 0-2-(trifl uoromethy Opropyl] -2-thiophenesulfonamide (Begacestat), MDL-28170,3,5-Bis(4-nitrophenoxy)benzoic acid (Compound W), 7-Amino-4-chloro-3-methoxy-1H-2-benzopyran (JLK6), (5S)-(tert-Butoxy carbonylamino)-6-phenyl-(4R)-hy droxy -(2R)-b enzy lhexanoy1)-L -leucy-L -phenylalaninamide (L-685,485), (R)-2-Fluoro-a-methyl[1,11-bipheny1]-4-acetic acid ((R)-Flurbiprofen; Flurizan), N-[(1S)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5H-dibenz[b,dIazepin-7-yl] amino] -1-methy1-2-oxoethyl] -3,5 -difluorobenzeneacetami de (Dibenzazepine; DBZ), N-[cis-4- [(4-ChlorophenyOsulfony11-4-(2,5-difluorophenyl)cy clohexyl] -1,1,1-trifluoromethanesulfonami de (MRK560), (2S)-2-[[(2S)-6,8-Difluoro-1,2,3,4-tetrahy dro-2-naphtha' enyl] amino] -N41 - [2- [(2,2-dimethylpropyl)amino] -1,1 -di methylethyl] -1H-imi dazol-4-yl]pentanamide dihydrobromide (PF3084014 hydrobromide) and 2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl] (2,5-difluorophenyl)amino] ethyl-5-fluorobenzenebutanoic acid (BMS299897).
Cyclin Dependent Kinase Inhibitors [00184] Cyclin dependent kinases (CDKs) are a family of sugar kinases first discovered for their role in regulating the cell cycle. They are also involved in regulating
- 48 -transcription, mRNA processing, and the differentiation of nerve cells. In many human cancers, CDKs are overactive or CDK-inhibiting proteins are not functional. CDK
inhibitors may be CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, and/or CDK9 inhibitors. In particular aspects, the CDK inhibitor is a CDK4/6 inhibitor.
[00185] CDK
inhibitors may include, but are not limited to, Palbociclib (PD-0332991) HC1, Roscovitine (Seliciclib, CYC202), SNS-032 (BMS-387032), Dinaciclib (SCH727965), Flavopiridol (Alvocidib), MSC2530818, JNJ-7706621, AZD5438, MK-(SCH 900776), PHA-793887, BS-181 HC1, A-674563, abemaciclib (LY2835219), BMS-265246, PHA-767491, or Milciclib (PHA-848125).
IV. Use of Photoreceptor Precursor Cells [00186]
Certain aspects provide a method to produce a PRP or PRP-enriched cell population which can be used for a number of important research, development, and commercial purposes.
[00187] In some aspects, the methods disclosed herein result in a cell population of at least or about 106, 107, 108, 5x108, 109, 1010 cells (or any range derivable therein) comprising at least or about 50% (for example, at least or about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or any range derivable therein) PRP cells.
[00188] In certain aspects, starting cells for the present methods may comprise the use of at least or about 104, 105, 106, 10, 108, 109, 1010, 1011, 1012, 1013 cells or any range derivable therein. The starting cell population may have a seeding density of at least or about 101, 102, 103, 104, 105, 106, 107, 108 cells/mL, or any range derivable therein.
[00189] The PRP cells or photoreceptor cells produced by the methods disclosed herein may be used in any methods and applications currently known in the art for PRP or photoreceptor cells. For example, a method of assessing a compound may be provided, comprising assaying a pharmacological or toxicological property of the compound on the PRP
or photoreceptor cell. There may also be provided a method of assessing a compound for an effect on a PRP cell, comprising: a) contacting the PRP cells provided herein with the compound; and b) assaying an effect of the compound on the PRP cells.
inhibitors may be CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, and/or CDK9 inhibitors. In particular aspects, the CDK inhibitor is a CDK4/6 inhibitor.
[00185] CDK
inhibitors may include, but are not limited to, Palbociclib (PD-0332991) HC1, Roscovitine (Seliciclib, CYC202), SNS-032 (BMS-387032), Dinaciclib (SCH727965), Flavopiridol (Alvocidib), MSC2530818, JNJ-7706621, AZD5438, MK-(SCH 900776), PHA-793887, BS-181 HC1, A-674563, abemaciclib (LY2835219), BMS-265246, PHA-767491, or Milciclib (PHA-848125).
IV. Use of Photoreceptor Precursor Cells [00186]
Certain aspects provide a method to produce a PRP or PRP-enriched cell population which can be used for a number of important research, development, and commercial purposes.
[00187] In some aspects, the methods disclosed herein result in a cell population of at least or about 106, 107, 108, 5x108, 109, 1010 cells (or any range derivable therein) comprising at least or about 50% (for example, at least or about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or any range derivable therein) PRP cells.
[00188] In certain aspects, starting cells for the present methods may comprise the use of at least or about 104, 105, 106, 10, 108, 109, 1010, 1011, 1012, 1013 cells or any range derivable therein. The starting cell population may have a seeding density of at least or about 101, 102, 103, 104, 105, 106, 107, 108 cells/mL, or any range derivable therein.
[00189] The PRP cells or photoreceptor cells produced by the methods disclosed herein may be used in any methods and applications currently known in the art for PRP or photoreceptor cells. For example, a method of assessing a compound may be provided, comprising assaying a pharmacological or toxicological property of the compound on the PRP
or photoreceptor cell. There may also be provided a method of assessing a compound for an effect on a PRP cell, comprising: a) contacting the PRP cells provided herein with the compound; and b) assaying an effect of the compound on the PRP cells.
- 49 -[00190] The PRP cells or cells derived from the PRP cells may be used for transplantation such as cell rescue therapy or whole tissue replacement therapy. The cells of the present embodiments may also be used to produce retinal disease models to study pathophysiology and for drug screening.
A. Test Compound Screening [00191] PRP
cells can be used commercially to screen for factors (such as solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of such cells and their various progeny. For example, test compounds may be chemical compounds, small molecules, polypeptides, growth factors, cytokines, or other biological agents.
[00192] In one embodiment, a method includes contacting a PRP cell with a test agent and determining if the test agent modulates activity or function of PRP
cells within the population. In some applications, screening assays are used for the identification of agents that modulate PRP cell proliferation, alter PRP cell differentiation, or affect cell viability. Screening assays may be performed in vitro or in vivo. Methods of screening and identifying ocular agents or PRP agents include those suitable for high-throughput screening. For example, PRP cells can be positioned or placed on a culture dish, flask, roller bottle or plate (e.g., a single multi-well dish or dish such as 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish), optionally at defined locations, for identification of potentially therapeutic molecules.
Libraries that can be screened include, for example, small molecule libraries, siRNA libraries, and adenoviral transfection vector libraries.
[00193]
Other screening applications relate to the testing of pharmaceutical compounds for their effect on retinal tissue maintenance or repair. Screening may be done either because the compound is designed to have a pharmacological effect on the cells, or because a compound designed to have effects elsewhere may have unintended side effects on cells of this tissue type.
B. Therapy and Transplantation [00194]
Other embodiments can also provide use of PRP cells to enhance ocular tissue maintenance and repair for any condition in need thereof, including retinal degeneration or significant injury. Retinal degeneration may be associated with age-related macular degeneration (AMD), Stargardt's macular dystrophy, retinitis pigmentosa, glaucoma, retinal
A. Test Compound Screening [00191] PRP
cells can be used commercially to screen for factors (such as solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of such cells and their various progeny. For example, test compounds may be chemical compounds, small molecules, polypeptides, growth factors, cytokines, or other biological agents.
[00192] In one embodiment, a method includes contacting a PRP cell with a test agent and determining if the test agent modulates activity or function of PRP
cells within the population. In some applications, screening assays are used for the identification of agents that modulate PRP cell proliferation, alter PRP cell differentiation, or affect cell viability. Screening assays may be performed in vitro or in vivo. Methods of screening and identifying ocular agents or PRP agents include those suitable for high-throughput screening. For example, PRP cells can be positioned or placed on a culture dish, flask, roller bottle or plate (e.g., a single multi-well dish or dish such as 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish), optionally at defined locations, for identification of potentially therapeutic molecules.
Libraries that can be screened include, for example, small molecule libraries, siRNA libraries, and adenoviral transfection vector libraries.
[00193]
Other screening applications relate to the testing of pharmaceutical compounds for their effect on retinal tissue maintenance or repair. Screening may be done either because the compound is designed to have a pharmacological effect on the cells, or because a compound designed to have effects elsewhere may have unintended side effects on cells of this tissue type.
B. Therapy and Transplantation [00194]
Other embodiments can also provide use of PRP cells to enhance ocular tissue maintenance and repair for any condition in need thereof, including retinal degeneration or significant injury. Retinal degeneration may be associated with age-related macular degeneration (AMD), Stargardt's macular dystrophy, retinitis pigmentosa, glaucoma, retinal
- 50 -vascular disease, viral infection of the eye, and other retinal/ocular disease. The photoreceptor precursor progenitor cells may comprise at least 50%, at least 75%, at least 85%, at least 95%, at least 99% or about 100% of the cells in the culture.
[00195] In another aspect, the disclosure provides a method of treatment of an individual in need thereof, comprising administering a composition comprising photoreceptor precursor cells to said individual. Said composition may be administered to the eye, subretinal space, or intravenously. Such individuals may have macular degeneration including age-related macular degeneration, and such macular degeneration may be early or late stage. Such individuals may have retinitis pigmentosa, Stargardt's disease, retinal dysplasia, retinal degeneration, diabetic retinopathy, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, glaucoma, or optic neuropathy.
[00196] To determine suitability of cell compositions for therapeutics administration, the cells can first be tested in a suitable animal model. In one aspect, the PRP
cells are evaluated for their ability to survive and maintain their phenotype in vivo. Cell compositions are administered to immunodeficient animals (e.g., nude mice or animals rendered immunodeficient chemically or by irradiation). Tissues are harvested after a period of growth, and assessed as to whether the pluripotent stem cell-derived cells are still present.
[00197] A
number of animals are available for testing of the suitability of the PRP cell compositions. For example, the Royal College of Surgeon's (RCS) rat is a well-known model of retinal dystrophy (Lund et al., 2006). In addition, PRP cell suitability and survival can be determined by transplantation (e.g., subcutaneous or subretinal) in Matrigel in immunodeficient animals such as NOG mice (Kanemura etal., 2014). Other models that may be used include, but are not limited to, the 5334ter rat model of retinal degeneration and the NIH Rowett nude (RNU) rat model.
[00198] The human PRP
cells described herein, or a pharmaceutical composition including these cells, can be used for the manufacture of a medicament to treat a condition in a patient in need thereof The PRP cells can be previously cryopreserved. In certain aspects, the disclosed PRP cells are derived from iPSCs, and thus can be used to provide "personalized medicine" for patients with eye diseases. In some embodiments, somatic cells obtained from patients can be genetically engineered to correct the disease-causing mutation, differentiated into PRP, and engineered to form a PRP tissue. This PRP tissue can be used to
[00195] In another aspect, the disclosure provides a method of treatment of an individual in need thereof, comprising administering a composition comprising photoreceptor precursor cells to said individual. Said composition may be administered to the eye, subretinal space, or intravenously. Such individuals may have macular degeneration including age-related macular degeneration, and such macular degeneration may be early or late stage. Such individuals may have retinitis pigmentosa, Stargardt's disease, retinal dysplasia, retinal degeneration, diabetic retinopathy, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, glaucoma, or optic neuropathy.
[00196] To determine suitability of cell compositions for therapeutics administration, the cells can first be tested in a suitable animal model. In one aspect, the PRP
cells are evaluated for their ability to survive and maintain their phenotype in vivo. Cell compositions are administered to immunodeficient animals (e.g., nude mice or animals rendered immunodeficient chemically or by irradiation). Tissues are harvested after a period of growth, and assessed as to whether the pluripotent stem cell-derived cells are still present.
[00197] A
number of animals are available for testing of the suitability of the PRP cell compositions. For example, the Royal College of Surgeon's (RCS) rat is a well-known model of retinal dystrophy (Lund et al., 2006). In addition, PRP cell suitability and survival can be determined by transplantation (e.g., subcutaneous or subretinal) in Matrigel in immunodeficient animals such as NOG mice (Kanemura etal., 2014). Other models that may be used include, but are not limited to, the 5334ter rat model of retinal degeneration and the NIH Rowett nude (RNU) rat model.
[00198] The human PRP
cells described herein, or a pharmaceutical composition including these cells, can be used for the manufacture of a medicament to treat a condition in a patient in need thereof The PRP cells can be previously cryopreserved. In certain aspects, the disclosed PRP cells are derived from iPSCs, and thus can be used to provide "personalized medicine" for patients with eye diseases. In some embodiments, somatic cells obtained from patients can be genetically engineered to correct the disease-causing mutation, differentiated into PRP, and engineered to form a PRP tissue. This PRP tissue can be used to
- 51 -replace the endogenous degenerated PRP of the same patient. Alternatively, iPSCs generated from a healthy donor or from HLA homozygous "super-donors" can be used.
[00199]
Various eye conditions may be treated or prevented by the introduction of the PRP cells obtained using the methods disclosed herein. The conditions include retinal diseases or disorders generally associated with retinal dysfunction or degradation, retinal injury, and/or loss of retinal pigment epithelium. Conditions that can be treated include, without limitation, degenerative diseases of the retina, such as Stargardt's macular dystrophy, retinitis pigmentosa, macular degeneration (such as age-related macular degeneration), glaucoma, and diabetic retinopathy. Additional conditions include Lebers congenital amaurosis, hereditary or acquired macular degeneration, Best disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, other dystrophies of photoreceptor cells, and retinal damage due to damage caused by any one of photic, laser, inflammatory, infectious, radiation, neovascular or traumatic injury. In certain embodiments, methods are provided for treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a composition comprising PRP
cells. These methods can include selecting a subject with one or more of these conditions, and administering a therapeutically effective amount of the PRP cells sufficient to treat the condition and/or ameliorate symptoms of the condition. The PRP cells may be transplanted in various formats.
For example, the PRP cells may be introduced into the target site in the form of cell suspension, or adhered onto a matrix, extracellular matrix or substrate such as a biodegradable polymer, as a monolayer, or a combination. The PRP cells may also be transplanted together (co-transplantation) with other retinal cells, such as with retinal pigment epithelium cells. In some embodiments, the PRP cells are produced from iPSCs from the subject to be treated, and thus are autologous. In other embodiments, the PRP cells are produced from an MHC-matched donor.
[00200]
Advantageously, the pharmaceutical preparations of the present disclosure may be used to compensate for a lack or diminution of photoreceptor cell function.
Examples of retinal dysfunction that can be treated by the retinal cell populations and methods of the invention include but are not limited to: photoreceptor degeneration (as occurs in, e.g., retinitis pigmentosa, cone dystrophies, cone-rod and/or rod-cone dystrophies, and macular degeneration); retina detachment and retinal trauma; photic lesions caused by laser or sunlight;
a macular hole; a macular edema; night blindness and color blindness; ischemic retinopathy as
[00199]
Various eye conditions may be treated or prevented by the introduction of the PRP cells obtained using the methods disclosed herein. The conditions include retinal diseases or disorders generally associated with retinal dysfunction or degradation, retinal injury, and/or loss of retinal pigment epithelium. Conditions that can be treated include, without limitation, degenerative diseases of the retina, such as Stargardt's macular dystrophy, retinitis pigmentosa, macular degeneration (such as age-related macular degeneration), glaucoma, and diabetic retinopathy. Additional conditions include Lebers congenital amaurosis, hereditary or acquired macular degeneration, Best disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, other dystrophies of photoreceptor cells, and retinal damage due to damage caused by any one of photic, laser, inflammatory, infectious, radiation, neovascular or traumatic injury. In certain embodiments, methods are provided for treating or preventing a condition characterized by retinal degeneration, comprising administering to a subject in need thereof an effective amount of a composition comprising PRP
cells. These methods can include selecting a subject with one or more of these conditions, and administering a therapeutically effective amount of the PRP cells sufficient to treat the condition and/or ameliorate symptoms of the condition. The PRP cells may be transplanted in various formats.
For example, the PRP cells may be introduced into the target site in the form of cell suspension, or adhered onto a matrix, extracellular matrix or substrate such as a biodegradable polymer, as a monolayer, or a combination. The PRP cells may also be transplanted together (co-transplantation) with other retinal cells, such as with retinal pigment epithelium cells. In some embodiments, the PRP cells are produced from iPSCs from the subject to be treated, and thus are autologous. In other embodiments, the PRP cells are produced from an MHC-matched donor.
[00200]
Advantageously, the pharmaceutical preparations of the present disclosure may be used to compensate for a lack or diminution of photoreceptor cell function.
Examples of retinal dysfunction that can be treated by the retinal cell populations and methods of the invention include but are not limited to: photoreceptor degeneration (as occurs in, e.g., retinitis pigmentosa, cone dystrophies, cone-rod and/or rod-cone dystrophies, and macular degeneration); retina detachment and retinal trauma; photic lesions caused by laser or sunlight;
a macular hole; a macular edema; night blindness and color blindness; ischemic retinopathy as
- 52 -caused by diabetes or vascular occlusion; retinopathy due to prematurity/premature birth;
infectious conditions, such as CMV, retinitis and toxoplasmosis; inflammatory conditions, such as the uveitidies; tumors, such as retinoblastoma and ocular melanoma;
and for the replacement of inner retinal neurons, which are affected in ocular neuropathies including glaucoma, traumatic optic neuropathy, and radiation optic neuropathy and retinopathy.
[00201] In one aspect, the cells can treat or alleviate the symptoms of retinitis pigmentosa in a patient in need of the treatment. In another aspect, the cells can treat or alleviate the symptoms of macular degeneration, such as age-related macular degeneration (wet or dry), Stargardt's disease, myopic macular degeneration or the like, in a patient in need of this treatment. For all of these treatments, the cells can be autologous or allogeneic to the patient.
In a further aspect, the cells of the present disclosure can be administered in combination with other treatments.
[00202] In some embodiments, the PRP cells can be used for autologous PRP
grafts to those subjects suitable for receiving regenerative medicine. The PRP
cells may be transplanted in combination with other retinal cells, such as with photoreceptors.
Transplantation of the PRP cells produced by the disclosed methods can be performed by various techniques known in the art. In accordance with one embodiment, the transplantation is performed via pars pana vitrectomy surgery followed by delivery of the cells through a small retinal opening into the sub-retinal space or by direct injection. The PRP
cells can be introduced into the target site in the form of cell suspension, cell aggregates, adhered onto a matrix, such as extracellular matrix, or provided on substrate such as a biodegradable polymer. The PRP
cells can also be transplanted together (co-transplantation) with other cells, such as PRP cells with retinal pigment epithelial (RPE) cells. Thus, a composition comprising PRP cells obtained by the methods disclosed herein is provided.
[00203] The PRP
cells, and optionally the photoreceptor cells differentiated therefrom, can be used to generate neurosensory retinal structures. For instance, the present disclosure contemplates the generation of multilayer cellular structures comprised of RPE cells and photoreceptor cells (or PRP cells). These structures can be used for drug screening, as models for diseases, or as or in a pharmaceutical preparation. In the latter case, the pharmaceutical preparation can be an RPE-photoreceptor graft, which may be disposed on a biocompatible solid support or matrix (preferably a bioresorbable matrix or support) that can be implanted like a "patch".
infectious conditions, such as CMV, retinitis and toxoplasmosis; inflammatory conditions, such as the uveitidies; tumors, such as retinoblastoma and ocular melanoma;
and for the replacement of inner retinal neurons, which are affected in ocular neuropathies including glaucoma, traumatic optic neuropathy, and radiation optic neuropathy and retinopathy.
[00201] In one aspect, the cells can treat or alleviate the symptoms of retinitis pigmentosa in a patient in need of the treatment. In another aspect, the cells can treat or alleviate the symptoms of macular degeneration, such as age-related macular degeneration (wet or dry), Stargardt's disease, myopic macular degeneration or the like, in a patient in need of this treatment. For all of these treatments, the cells can be autologous or allogeneic to the patient.
In a further aspect, the cells of the present disclosure can be administered in combination with other treatments.
[00202] In some embodiments, the PRP cells can be used for autologous PRP
grafts to those subjects suitable for receiving regenerative medicine. The PRP
cells may be transplanted in combination with other retinal cells, such as with photoreceptors.
Transplantation of the PRP cells produced by the disclosed methods can be performed by various techniques known in the art. In accordance with one embodiment, the transplantation is performed via pars pana vitrectomy surgery followed by delivery of the cells through a small retinal opening into the sub-retinal space or by direct injection. The PRP
cells can be introduced into the target site in the form of cell suspension, cell aggregates, adhered onto a matrix, such as extracellular matrix, or provided on substrate such as a biodegradable polymer. The PRP
cells can also be transplanted together (co-transplantation) with other cells, such as PRP cells with retinal pigment epithelial (RPE) cells. Thus, a composition comprising PRP cells obtained by the methods disclosed herein is provided.
[00203] The PRP
cells, and optionally the photoreceptor cells differentiated therefrom, can be used to generate neurosensory retinal structures. For instance, the present disclosure contemplates the generation of multilayer cellular structures comprised of RPE cells and photoreceptor cells (or PRP cells). These structures can be used for drug screening, as models for diseases, or as or in a pharmaceutical preparation. In the latter case, the pharmaceutical preparation can be an RPE-photoreceptor graft, which may be disposed on a biocompatible solid support or matrix (preferably a bioresorbable matrix or support) that can be implanted like a "patch".
- 53 -[00204] To further illustrate, the biocompatible support for the cells can be a biodegradable synthetic, such as polyester, film support for retinal progenitor cells. The biodegradable polyester can be any biodegradable polyester suitable for use as a substrate or scaffold for supporting the proliferation and differentiation of retinal progenitor cells. The polyester should be capable of forming a thin film, preferably a micro-textured film, and should be biodegradable if used for tissue or cell transplantation. Suitable biodegradable polyesters for use in the invention include polylactic acid (PLA), polylactides, polyhydroxyalkanoates, both homopolymers and co-polymers, such as polyhydoxybutyrate (PHB), polyhydroxybutyrate co-hydroxyvalerate (PHBV), polyhydroxybutyrate co-hydroxyhexanote (PHBHx), polyhydroxybutyrate co- hydroxyoctonoate (PHBO) and polyhydroxybutyrate co-hydroxyoctadecanoate (PHBOd), polycaprolactone (PCL), polyesteramide (PEA), aliphatic copolyesters, such as polybutylene succinate (PBS) and polybutylene succinate/adipate (PBSA), aromatic copolyesters. Both high and low molecular weight polyesters, substituted and unsubstituted polyester, block, branched or random, and polyester mixtures and blends can be used. Preferably the biodegradable polyester is polycaprolactone (PCL).
[00205]
Pharmaceutical compositions of the PRP cells produced by the methods disclosed herein. These compositions can include at least about 1 x 103 PRP
cells, about 1 x 104 PRP cells, about 1 x 105 PRP cells, about 1 x 106 PRP cells, about 1 x 107 PRP cells, about 1 x 108 PRP cells, or about 1 x 109 PRP cells. In certain embodiments, the compositions are substantially purified (with respect to non-PRP cells) preparations comprising differentiated PRP cells produced by the methods disclosed herein. Compositions are also provided that include a scaffold, such as a polymeric carrier and/or an extracellular matrix, and an effective amount of the PRP cells produced by the methods disclosed herein. For example, the cells are provided as a monolayer of cells. The matrix material is generally physiologically acceptable and suitable for use in in vivo applications. For example, the physiologically acceptable materials include, but are not limited to, solid matrix materials that are absorbable and/or non-absorbable, such as small intestine submucosa (SIS), crosslinked or non-crosslinked alginate, hydrocolloid, foams, collagen gel, collagen sponge, polyglycolic acid (PGA) mesh, fleeces and bioadhesives.
[00206] Suitable polymeric carriers also include porous meshes or sponges formed of synthethic or natural polymers, as well as polymer solutions. For example, the matrix is a polymeric mesh or sponge, or a polymeric hydrogel. Natural polymers that can be used
[00205]
Pharmaceutical compositions of the PRP cells produced by the methods disclosed herein. These compositions can include at least about 1 x 103 PRP
cells, about 1 x 104 PRP cells, about 1 x 105 PRP cells, about 1 x 106 PRP cells, about 1 x 107 PRP cells, about 1 x 108 PRP cells, or about 1 x 109 PRP cells. In certain embodiments, the compositions are substantially purified (with respect to non-PRP cells) preparations comprising differentiated PRP cells produced by the methods disclosed herein. Compositions are also provided that include a scaffold, such as a polymeric carrier and/or an extracellular matrix, and an effective amount of the PRP cells produced by the methods disclosed herein. For example, the cells are provided as a monolayer of cells. The matrix material is generally physiologically acceptable and suitable for use in in vivo applications. For example, the physiologically acceptable materials include, but are not limited to, solid matrix materials that are absorbable and/or non-absorbable, such as small intestine submucosa (SIS), crosslinked or non-crosslinked alginate, hydrocolloid, foams, collagen gel, collagen sponge, polyglycolic acid (PGA) mesh, fleeces and bioadhesives.
[00206] Suitable polymeric carriers also include porous meshes or sponges formed of synthethic or natural polymers, as well as polymer solutions. For example, the matrix is a polymeric mesh or sponge, or a polymeric hydrogel. Natural polymers that can be used
- 54 -include proteins such as collagen, albumin, and fibrin; and polysaccharides such as alginate and polymers of hyaluronic acid. Synthetic polymers include both biodegradable and non-biodegradable polymers. For example, biodegradable polymers include polymers of hydroxy acids such as polyactic acid (PLA), polyglycolic acid (PGA) and polylactic acid-glycolic acid (PGLA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, and polyvinyl alcohols.
[00207]
Polymers that can form ionic or covalently crosslinked hydrogels which are malleable can be used. A hydrogel is a substance formed when an organic polymer (natural or synthetic) is cross- linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block copolymers such as PLURON1CSTM or TETRON1CSTm, polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or H, respectively. Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen.
[00208] The pharmaceutical compositions can be optionally packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution of PRP cell function to improve a disease or abnormality of the retinal tissue.
In some embodiments, the PRP cells produced by the disclosed methods may be used to replace degenerated photoreceptor cells of a subject in need therein.
C. Distribution for Commercial, Therapeutic, and Research Purposes [00209] In some embodiments, a reagent system is provided that includes a set or combination of cells comprising a PRP or PRP-enriched cell population that exists at any time during manufacture, distribution or use. The cell sets comprise any combination of the cell population described herein in combination with undifferentiated pluripotent stem cells or other differentiated cell types, often sharing the same genome. Each cell type may be packaged together, or in separate containers in the same facility, or at different locations, at the same or different times, under control of the same entity or different entities sharing a business relationship.
[00207]
Polymers that can form ionic or covalently crosslinked hydrogels which are malleable can be used. A hydrogel is a substance formed when an organic polymer (natural or synthetic) is cross- linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block copolymers such as PLURON1CSTM or TETRON1CSTm, polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or H, respectively. Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen.
[00208] The pharmaceutical compositions can be optionally packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution of PRP cell function to improve a disease or abnormality of the retinal tissue.
In some embodiments, the PRP cells produced by the disclosed methods may be used to replace degenerated photoreceptor cells of a subject in need therein.
C. Distribution for Commercial, Therapeutic, and Research Purposes [00209] In some embodiments, a reagent system is provided that includes a set or combination of cells comprising a PRP or PRP-enriched cell population that exists at any time during manufacture, distribution or use. The cell sets comprise any combination of the cell population described herein in combination with undifferentiated pluripotent stem cells or other differentiated cell types, often sharing the same genome. Each cell type may be packaged together, or in separate containers in the same facility, or at different locations, at the same or different times, under control of the same entity or different entities sharing a business relationship.
- 55 -[00210]
Pharmaceutical compositions may optionally be packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution of PRP cell function to improve a disease or injury of the ocular tissue.
V. Kits [00211] In some embodiments, a kit that can include, for example, one or more media and components for the production of PRP cells is provided. Such formulations may comprise a cocktail of retinal differentiation and/or trophic factors, in a form suitable for combining with photoreceptor precursor or photoreceptor cells. The reagent system may be packaged either in aqueous media or in lyophilized form, where appropriate.
The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted.
Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits also will typically include a means for containing the kit component(s) in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. The kit can also include instructions for use, such as in printed or electronic format, such as digital format.
VI. Examples [00212] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Pharmaceutical compositions may optionally be packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution of PRP cell function to improve a disease or injury of the ocular tissue.
V. Kits [00211] In some embodiments, a kit that can include, for example, one or more media and components for the production of PRP cells is provided. Such formulations may comprise a cocktail of retinal differentiation and/or trophic factors, in a form suitable for combining with photoreceptor precursor or photoreceptor cells. The reagent system may be packaged either in aqueous media or in lyophilized form, where appropriate.
The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted.
Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits also will typically include a means for containing the kit component(s) in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. The kit can also include instructions for use, such as in printed or electronic format, such as digital format.
VI. Examples [00212] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
- 56 -Example 1 ¨ Preparation of Starting Pluripotent Stem Cell Population [00213] A
method was developed for the differentiation of iPSCs into different stages of photoreceptor precursors (PRPs) (FIG. 1). Briefly, a population of retinal progenitor cells (RPCs) is derived from iPSCs which are then further differentiated to neural retinal progenitors (NRPs) and then PRP cells.
[00214]
First, the iPSCs were grown without mouse or human feeder layers in fully defined-culture medium, such as ESSENTIAL 8TM (E8TM) medium, on a plate coated by vitronectin. The vitronectin stock was diluted 1:200 in DPBS without calcium or magnesium and the culture plates were coated with the diluted vitronectin solution and incubated at room temperature for about 1 hour. The iPSCs were split when they were pre-confluent and not allowed to overgrow to prevent unhealthy and/or differentiated cells.
[00215] To derive RPCs, the iPSCs were first dissociated into a single cell suspension. To obtain the single cell suspension, the cells were washed with DPBS (without calcium and magnesium) and incubated in a cell dissociation enzyme such as TRYPLETm for about 10 min at 37 C. The cells were then detached by pipetting with a serological pipet and the cell suspension was collected in a conical tube. If the cells did not detach with gently pipetting, the cultures were incubated longer, such as 2-3 additional minutes.
To collect all cells, the culture vessel was washed with room temperature E8TM medium, and the medium was then added to the tube containing the cell suspension. In addition, Blebbistatin (e.g. 2.5 [tM) was added to the E8TM Medium to increase PSC survival after dissociation into single cells while the cells are not adhered to a culture vessel. To collect the cells, they were centrifuged at 400xg for about 5 minutes, the supernatant was aspirated and the cells were resuspended in an appropriate volume of E8TM medium.
[00216] To efficiently differentiate PRP cells from the single cell iPSCs, the input density of the single cell iPSCs was accurately counted by an automated cell counter such as VICELLTM and diluted to a cell suspension of about 1x105 cells/mL in room temperature E8TM medium. Once the single cell suspension of iPSCs was obtained at a known cell density (e.g., by hemocytometer), the cells were plated in an appropriate culture vessel such as a 6-well plate coated with vitronectin. The cells were seeded at a cell density of about 200,000 cells per well and placed in a humidified incubator at 5% CO2 and 37 C. After about 18-24 hours, the medium was aspirated and fresh E8TM medium was added to the culture. The cells were
method was developed for the differentiation of iPSCs into different stages of photoreceptor precursors (PRPs) (FIG. 1). Briefly, a population of retinal progenitor cells (RPCs) is derived from iPSCs which are then further differentiated to neural retinal progenitors (NRPs) and then PRP cells.
[00214]
First, the iPSCs were grown without mouse or human feeder layers in fully defined-culture medium, such as ESSENTIAL 8TM (E8TM) medium, on a plate coated by vitronectin. The vitronectin stock was diluted 1:200 in DPBS without calcium or magnesium and the culture plates were coated with the diluted vitronectin solution and incubated at room temperature for about 1 hour. The iPSCs were split when they were pre-confluent and not allowed to overgrow to prevent unhealthy and/or differentiated cells.
[00215] To derive RPCs, the iPSCs were first dissociated into a single cell suspension. To obtain the single cell suspension, the cells were washed with DPBS (without calcium and magnesium) and incubated in a cell dissociation enzyme such as TRYPLETm for about 10 min at 37 C. The cells were then detached by pipetting with a serological pipet and the cell suspension was collected in a conical tube. If the cells did not detach with gently pipetting, the cultures were incubated longer, such as 2-3 additional minutes.
To collect all cells, the culture vessel was washed with room temperature E8TM medium, and the medium was then added to the tube containing the cell suspension. In addition, Blebbistatin (e.g. 2.5 [tM) was added to the E8TM Medium to increase PSC survival after dissociation into single cells while the cells are not adhered to a culture vessel. To collect the cells, they were centrifuged at 400xg for about 5 minutes, the supernatant was aspirated and the cells were resuspended in an appropriate volume of E8TM medium.
[00216] To efficiently differentiate PRP cells from the single cell iPSCs, the input density of the single cell iPSCs was accurately counted by an automated cell counter such as VICELLTM and diluted to a cell suspension of about 1x105 cells/mL in room temperature E8TM medium. Once the single cell suspension of iPSCs was obtained at a known cell density (e.g., by hemocytometer), the cells were plated in an appropriate culture vessel such as a 6-well plate coated with vitronectin. The cells were seeded at a cell density of about 200,000 cells per well and placed in a humidified incubator at 5% CO2 and 37 C. After about 18-24 hours, the medium was aspirated and fresh E8TM medium was added to the culture. The cells were
- 57 -cultured in the E8TM medium for about 2 days after seeding for proper adherence and iPSC
expansion.
Example 2¨ Differentiation of iPSCs into RPCs [00217]
Once the single-cell iPSCs seeded at the appropriate cell density were cultured for about 2 days as in Example 1, they were cultured in various differentiation media for deriving RPCs. The E8TM medium was aspirated and room temperature Retinal Induction Medium (RIM) (e.g., Table 1) was added. Briefly, the RIM comprised DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, the RIM comprised a WNT pathway inhibitor, a BMP pathway inhibitor, a TGF13 pathway inhibitor and insulin growth factor 1 (IGF1). Each day the media was aspirated and fresh RIM was added to the cells. The cells were cultured in the RIM for about two days to generate anterior neuroectoderm cells.
[00218] The cells were then cultured in Retinal Differentiation Medium 1 (RD1) for about one to four days, particularly about two days. Briefly, the RD1 (Table 1) comprised DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, the RD1 comprised a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), a TGFP
pathway inhibitor (e.g., SB431542), and a MEK inhibitor (e.g., PD325901), and IGF-1. The concentration of the WNT pathway inhibitor, BMP pathway inhibitor and TGFP
pathway inhibitor was ten times higher in the RDM as compared to the RIM. Each day the media was aspirated and room temperature RD1 was added to the cells to produce differentiated retinal cells. In particular, the BMP inhibitor may be removed after the first few days, such as after 2 days, to enhance expression of VSX2 in the PRP cells. The RDM1 media may be replaced with RD2 (Table 1) which does not comprise the BMP inhibitor, such as LDN193189.
The cells may be cultured in the RD2 for about five to ten days, such as about seven days.
[00219] To derive NRP cells, the cells were then cultured in Retinal Maturation Medium (RM1 or RM2) (Table 1) for about five days to differentiate the RPCs cells to NRPs.
The RM1 comprised DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM
NEAA, sodium pyruvate, N-2 supplement, B-27 supplement and ascorbic acid. In addition, the RM1 comprised Nicotinamide, but was free of Activin A to prevent differentiation towards RPE cells. The RM1 media may further comprise a y-secretase inhibitor, such as DAPT. The
expansion.
Example 2¨ Differentiation of iPSCs into RPCs [00217]
Once the single-cell iPSCs seeded at the appropriate cell density were cultured for about 2 days as in Example 1, they were cultured in various differentiation media for deriving RPCs. The E8TM medium was aspirated and room temperature Retinal Induction Medium (RIM) (e.g., Table 1) was added. Briefly, the RIM comprised DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, the RIM comprised a WNT pathway inhibitor, a BMP pathway inhibitor, a TGF13 pathway inhibitor and insulin growth factor 1 (IGF1). Each day the media was aspirated and fresh RIM was added to the cells. The cells were cultured in the RIM for about two days to generate anterior neuroectoderm cells.
[00218] The cells were then cultured in Retinal Differentiation Medium 1 (RD1) for about one to four days, particularly about two days. Briefly, the RD1 (Table 1) comprised DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, the RD1 comprised a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), a TGFP
pathway inhibitor (e.g., SB431542), and a MEK inhibitor (e.g., PD325901), and IGF-1. The concentration of the WNT pathway inhibitor, BMP pathway inhibitor and TGFP
pathway inhibitor was ten times higher in the RDM as compared to the RIM. Each day the media was aspirated and room temperature RD1 was added to the cells to produce differentiated retinal cells. In particular, the BMP inhibitor may be removed after the first few days, such as after 2 days, to enhance expression of VSX2 in the PRP cells. The RDM1 media may be replaced with RD2 (Table 1) which does not comprise the BMP inhibitor, such as LDN193189.
The cells may be cultured in the RD2 for about five to ten days, such as about seven days.
[00219] To derive NRP cells, the cells were then cultured in Retinal Maturation Medium (RM1 or RM2) (Table 1) for about five days to differentiate the RPCs cells to NRPs.
The RM1 comprised DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM
NEAA, sodium pyruvate, N-2 supplement, B-27 supplement and ascorbic acid. In addition, the RM1 comprised Nicotinamide, but was free of Activin A to prevent differentiation towards RPE cells. The RM1 media may further comprise a y-secretase inhibitor, such as DAPT. The
- 58 -RM2 may further comprise a TGFr3 inhibitor, such as SB431542, and/or bFGF. The medium was changed daily with room temperature RM1. The RPCs were then cultured in photoreceptor precursor induction mediun (FDSC) medium from Days 15-18 to produce NRP cells.
To analyze the NRP cells, they were thawed into post-thaw medium and assayed for expression of PAX6 and VSX2 (FIG. 14). The NRP cells were found to be almost 100% positive for PAX6, about 90% positive for PMEL17, and about 80% positive for VSX2 (FIG. 14).
Example 3¨ Differentiation of RPCs to PRP Cells [00220] To complete the differentiation process to PRP cells, the RPC cells of Example 2 were cultured in FDSC medium (Table 1). Briefly, the FDSC Medium comprises DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement and ascorbic acid. In addition, the FDSC Medium may comprise basic FGF, DAPT, SB431542, and CKI-7 to differentiate the NRPs towards PRP
cells. The cells were cultured in the FDSC medium for about ten to twenty days, particularly fifteen days. The PRP cells were analyzed for expression of the markers Tuj 1/Nestin and RCVRN (FIGS. 4A-4C).
[00221] At this stage, the derived PRP cells can be dissociated using TRYPLETm and cryopreserved in xenofree CS10 medium. Alternatively, the derived PRP
cells can be cultured in medium comprising ROCK inhibitor or blebbistatin to promote aggregate formation for one to five days, such as three days, to promote better cell survival and transplantation.
Thus, the presently disclosed methods provide PRP cells from pluripotent cells that can be consistently reproduced at a large scale for clinical applications.
Example 4¨ Production of Optic Vesicles [00222] The RPC cells of Example 2 were cultured as aggregates in RM1 or RM2 media for an extended duration to produce optic vesicles. Blebbistatin was used to promote aggregate formation. The RM1 media comprised DMEM/F12, KnockOut serum replacement, sodium pyruvate, ascorbic acid, and nicotinamide. The optic vesicles were evaluated for marker expression at Day 30, Day 40, Day 50, and Day 60. There was an increase in RCVRN expression from 3.3% at Day 40 to over 30% at Day 78 (FIG. 12D).
Example 5¨ Alternative Method for Production of PRP Cells
To analyze the NRP cells, they were thawed into post-thaw medium and assayed for expression of PAX6 and VSX2 (FIG. 14). The NRP cells were found to be almost 100% positive for PAX6, about 90% positive for PMEL17, and about 80% positive for VSX2 (FIG. 14).
Example 3¨ Differentiation of RPCs to PRP Cells [00220] To complete the differentiation process to PRP cells, the RPC cells of Example 2 were cultured in FDSC medium (Table 1). Briefly, the FDSC Medium comprises DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement and ascorbic acid. In addition, the FDSC Medium may comprise basic FGF, DAPT, SB431542, and CKI-7 to differentiate the NRPs towards PRP
cells. The cells were cultured in the FDSC medium for about ten to twenty days, particularly fifteen days. The PRP cells were analyzed for expression of the markers Tuj 1/Nestin and RCVRN (FIGS. 4A-4C).
[00221] At this stage, the derived PRP cells can be dissociated using TRYPLETm and cryopreserved in xenofree CS10 medium. Alternatively, the derived PRP
cells can be cultured in medium comprising ROCK inhibitor or blebbistatin to promote aggregate formation for one to five days, such as three days, to promote better cell survival and transplantation.
Thus, the presently disclosed methods provide PRP cells from pluripotent cells that can be consistently reproduced at a large scale for clinical applications.
Example 4¨ Production of Optic Vesicles [00222] The RPC cells of Example 2 were cultured as aggregates in RM1 or RM2 media for an extended duration to produce optic vesicles. Blebbistatin was used to promote aggregate formation. The RM1 media comprised DMEM/F12, KnockOut serum replacement, sodium pyruvate, ascorbic acid, and nicotinamide. The optic vesicles were evaluated for marker expression at Day 30, Day 40, Day 50, and Day 60. There was an increase in RCVRN expression from 3.3% at Day 40 to over 30% at Day 78 (FIG. 12D).
Example 5¨ Alternative Method for Production of PRP Cells
- 59 -[00223] The RPC cells of Example 2 were cultured as aggregates in RM1 or RM2 media (Table 1) for about 40 to 70 days. Blebbistatin was used during aggregate formation. The RM2 media comprises DAPT in addition to the components of the RM1 media.
The cells remained in aggregates until approximately day 68, at which point they were dissociated, treated with BENZONASEO and plated for 7 days in RM2 media to generate Hybrid PRP cells. These Hybrid PRP cells displayed a phenotype similar to the PRP cells of Example 3. After magnetic-activated cell sorting (MACS) purification, the Hybrid PRP cell population was over 97% positive for TUBB3 while negative (<3%) for NESTIN and almost 80% positive for RCVRN (FIG. 10).
Example 6¨ Cryopreservation of NRPs or PRPS
[00224] For the cryopreservation of the differentiated NRPs or PRPs, the medium was aspirated and the cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS). The cells were then incubated with a cell dissociation enzyme and the cell suspension was pipetted into a conical tube. The cells were centrifuged, the supernatant aspirated and the cells resuspended in room temperature medium. The cell suspension was then filtered through a STERIFLIPO cell strainer (20 p.m) and the cells were counted. Next, the cells were centrifuged and resuspended at an appropriate density (e.g. 1x107cells/mL) in cold CryoStor0 CS10. The cell suspension was aliquoted into pre-labeled cryovials which were placed in a cold freezing container and transferred to a -80 C freezer for 12-24 hours. The vials were then transferred to liquid nitrogen for storage.
[00225]
Alternatively, the cells may be cryopreserved as aggregates instead of dissociation into the single cell suspension. For example, following CliniMACS
enrichment of D75 PRP, single cells were allowed to re-aggregate in tissue culture flasks for two days in minimal medium (RMN). Aggregates were pooled at D77 and a sample aliquot was obtained for cell counts. Following a series of washes, aggregates were resuspended in CryoSTOR CS10 Freeze Medium and 1 ml of the aggregate suspensions were transferred to liquid nitrogen storage vials at 25x10^6 aggregated cell products/vial.
[00226]
Morphologically, thawed aggregates remaining in culture were not different than aggregates formed from single cells 2 days post-thaw. The thawed aggregates had 67% cell viability compared to 37% for thawed single cells. Four conditions were tested for RCVRN (on-target PRP marker), CHX10 (early eye field cells), RCVRN+, CHX10 (off-
The cells remained in aggregates until approximately day 68, at which point they were dissociated, treated with BENZONASEO and plated for 7 days in RM2 media to generate Hybrid PRP cells. These Hybrid PRP cells displayed a phenotype similar to the PRP cells of Example 3. After magnetic-activated cell sorting (MACS) purification, the Hybrid PRP cell population was over 97% positive for TUBB3 while negative (<3%) for NESTIN and almost 80% positive for RCVRN (FIG. 10).
Example 6¨ Cryopreservation of NRPs or PRPS
[00224] For the cryopreservation of the differentiated NRPs or PRPs, the medium was aspirated and the cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS). The cells were then incubated with a cell dissociation enzyme and the cell suspension was pipetted into a conical tube. The cells were centrifuged, the supernatant aspirated and the cells resuspended in room temperature medium. The cell suspension was then filtered through a STERIFLIPO cell strainer (20 p.m) and the cells were counted. Next, the cells were centrifuged and resuspended at an appropriate density (e.g. 1x107cells/mL) in cold CryoStor0 CS10. The cell suspension was aliquoted into pre-labeled cryovials which were placed in a cold freezing container and transferred to a -80 C freezer for 12-24 hours. The vials were then transferred to liquid nitrogen for storage.
[00225]
Alternatively, the cells may be cryopreserved as aggregates instead of dissociation into the single cell suspension. For example, following CliniMACS
enrichment of D75 PRP, single cells were allowed to re-aggregate in tissue culture flasks for two days in minimal medium (RMN). Aggregates were pooled at D77 and a sample aliquot was obtained for cell counts. Following a series of washes, aggregates were resuspended in CryoSTOR CS10 Freeze Medium and 1 ml of the aggregate suspensions were transferred to liquid nitrogen storage vials at 25x10^6 aggregated cell products/vial.
[00226]
Morphologically, thawed aggregates remaining in culture were not different than aggregates formed from single cells 2 days post-thaw. The thawed aggregates had 67% cell viability compared to 37% for thawed single cells. Four conditions were tested for RCVRN (on-target PRP marker), CHX10 (early eye field cells), RCVRN+, CHX10 (off-
- 60 -target bipolar cells), Ki67 (off-target proliferating cell) and Pax6 (off-target neuroectoderm/early eye field) expression: Cryopreserved single cells thawed and allowed to re-aggregate for 2 days in RMN medium (SC D77 2d post-thaw aggregates); single cells following enrichment allowed to aggregate for 2 days in RMN, without cryopreservation (D77 pre-cryo cultured aggregates); cryopreserved aggregates immediately post-thaw (D77 aggregates at thaw); and cryopreserved aggregates thawed and cultured 2 days in RMN
medium (D79 thawed aggregates cultured 2d). There was no difference in RCVRN
expression among the four conditions. Off-target markers for early eye field (CHX10+ and Pax6+) remained low in all conditions and proliferative cells (Ki67+) were negligible.
[00227] Table 2. Mean percent on-target and off-target markers in aggregated single cells versus thawed cryopreserved aggregates (reported as mean SEM).
RCVRN CHX10 RCVRN+CHX10 Ki67% Pax6 %
Sc D77 2d post- 94.6 0.03 4.6 0.03 2.5 0.02 0.2 0.001 4.0 0.01 thaw aggregates D77 pre-cryo 92.3 0.02 5.2 0.02 3.9 0.01 0.1 0.001 3.4 0.01 cultured aggregates D77 aggregates at 95.2 0.02 3.7 0.01 2.4 0.01 0.1 0.000 3.1 0.01 thaw D79 thawed 91.4 0.03 4.4 0.01 2.4 0.01 0.5 0.002 5.1 0.02 aggregates cultured 2d [00228]
Thus, it was shown that not only can cryopreserve aggregates be successfully cryopreserved, but the enriched PRPs are not compromised during the cryopreservation or the thawing stages. This was evident by the similar high expression of the on-target PRP marker recoverin. Similarly, off-target markers and proliferating cell makers remained equally low throughout all conditions. Cryopreserving aggregates allows the product to go from the lab to the patient with minimal "in between" handing, as the transplanted product will be in aggregate form. The elimination of the 2 day "in between" culture step minimizes concerns of contamination and cell loss.
Example 7¨ MACS Purification of PRP Cells [00229] The population of PRP cells can have residual contaminating non-PRP
cells such as RPE cells or other non-neuronal cell types (collectively referred to as the "contaminating cells"), all of which can be separated and removed to yield a PRP-enriched cell
medium (D79 thawed aggregates cultured 2d). There was no difference in RCVRN
expression among the four conditions. Off-target markers for early eye field (CHX10+ and Pax6+) remained low in all conditions and proliferative cells (Ki67+) were negligible.
[00227] Table 2. Mean percent on-target and off-target markers in aggregated single cells versus thawed cryopreserved aggregates (reported as mean SEM).
RCVRN CHX10 RCVRN+CHX10 Ki67% Pax6 %
Sc D77 2d post- 94.6 0.03 4.6 0.03 2.5 0.02 0.2 0.001 4.0 0.01 thaw aggregates D77 pre-cryo 92.3 0.02 5.2 0.02 3.9 0.01 0.1 0.001 3.4 0.01 cultured aggregates D77 aggregates at 95.2 0.02 3.7 0.01 2.4 0.01 0.1 0.000 3.1 0.01 thaw D79 thawed 91.4 0.03 4.4 0.01 2.4 0.01 0.5 0.002 5.1 0.02 aggregates cultured 2d [00228]
Thus, it was shown that not only can cryopreserve aggregates be successfully cryopreserved, but the enriched PRPs are not compromised during the cryopreservation or the thawing stages. This was evident by the similar high expression of the on-target PRP marker recoverin. Similarly, off-target markers and proliferating cell makers remained equally low throughout all conditions. Cryopreserving aggregates allows the product to go from the lab to the patient with minimal "in between" handing, as the transplanted product will be in aggregate form. The elimination of the 2 day "in between" culture step minimizes concerns of contamination and cell loss.
Example 7¨ MACS Purification of PRP Cells [00229] The population of PRP cells can have residual contaminating non-PRP
cells such as RPE cells or other non-neuronal cell types (collectively referred to as the "contaminating cells"), all of which can be separated and removed to yield a PRP-enriched cell
- 61 -population. The contaminating cells can be removed from the culture by various methodologies, such as, for example, Magnetic Activated Cell Sorting (MACS ), Fluorescent Activated Cell Sorting (FACS), or single cell sorting by positive selection and/or negative selection. The MACS methodology, which is known in the art to separate various cell populations depending on their surface antigens, was used to separate the contaminating cells from the desired PRP cells.
[00230]
Positive MACS selection for PRP cells can include isolation of cells which express the neuronal marker CD171. In order to carry out the positive MACS selection for PRP cells, the total population of PRP cells from Example 3 or 6 were dissociated into a single cell suspension. The medium was aspirated, the cells were washed with DPBS, and TRYPLE was added to dissociate the cells. The cells were then collected and centrifuged.
The cells were washed in PRP medium and filtered using a 20[tm steriflip cell strainer. The cell suspension was counted using ViCell and MACS buffer was added to the cell suspension at 1x107 cells/mL. Next, the cell suspension was stained with the primary antibody CD171-biotin (Miltenyi) at a 1:25 dilution. After incubation, 20 mL of MACS buffer was added and the cells were centrifuged at 400xg for 5 min. The cell pellet was resuspended in 20 mL MACS
buffer, vigorously mixed, and centrifuged at 400xg for 5 min to remove any unbound antibody.
The cell pellet was resuspended in MACS buffer (e.g., at 1.11x108 cells/mL), microbeads coated with the diluted (1:10) secondary antibody (e.g., anti-Biotin) were added, and the cells were incubated at 4 C for 20 min. After incubation, the cells were washed with MACS buffer to remove unbound microbeads and up to 1.25x108 cells were resuspended in 500 [IL MACS
buffer. The cell suspension was transferred to a LS column placed in a strong magnetic field and the cells expressing the antigen CD171 attached to the microbeads remained in the column.
The LS column was washed two times with MACS buffer. The enriched cell population was then flushed from the LS column and evaluated for post-sort purity analysis.
Images of cell staining for RCVRN are shown in FIGS. 3 and 9 depicting that the MACS
enrichment results in a significant enrichment of PRP cells.
[00231]
Further studies were performed to identify additional surface proteins for the enrichment of PRP cells. Surface protein evaluation using Miltenyi Marker Screen plate identified SUSD2 as a surface protein target to enrich for photoreceptor precursor (PRP) product. Sushi Domain-Containing Protein-2 (SUSD2) is a type I transmembrane protein that facilitates cell-cell and cell-matrix adhesion. Overexpression of SUSD2 is observed in
[00230]
Positive MACS selection for PRP cells can include isolation of cells which express the neuronal marker CD171. In order to carry out the positive MACS selection for PRP cells, the total population of PRP cells from Example 3 or 6 were dissociated into a single cell suspension. The medium was aspirated, the cells were washed with DPBS, and TRYPLE was added to dissociate the cells. The cells were then collected and centrifuged.
The cells were washed in PRP medium and filtered using a 20[tm steriflip cell strainer. The cell suspension was counted using ViCell and MACS buffer was added to the cell suspension at 1x107 cells/mL. Next, the cell suspension was stained with the primary antibody CD171-biotin (Miltenyi) at a 1:25 dilution. After incubation, 20 mL of MACS buffer was added and the cells were centrifuged at 400xg for 5 min. The cell pellet was resuspended in 20 mL MACS
buffer, vigorously mixed, and centrifuged at 400xg for 5 min to remove any unbound antibody.
The cell pellet was resuspended in MACS buffer (e.g., at 1.11x108 cells/mL), microbeads coated with the diluted (1:10) secondary antibody (e.g., anti-Biotin) were added, and the cells were incubated at 4 C for 20 min. After incubation, the cells were washed with MACS buffer to remove unbound microbeads and up to 1.25x108 cells were resuspended in 500 [IL MACS
buffer. The cell suspension was transferred to a LS column placed in a strong magnetic field and the cells expressing the antigen CD171 attached to the microbeads remained in the column.
The LS column was washed two times with MACS buffer. The enriched cell population was then flushed from the LS column and evaluated for post-sort purity analysis.
Images of cell staining for RCVRN are shown in FIGS. 3 and 9 depicting that the MACS
enrichment results in a significant enrichment of PRP cells.
[00231]
Further studies were performed to identify additional surface proteins for the enrichment of PRP cells. Surface protein evaluation using Miltenyi Marker Screen plate identified SUSD2 as a surface protein target to enrich for photoreceptor precursor (PRP) product. Sushi Domain-Containing Protein-2 (SUSD2) is a type I transmembrane protein that facilitates cell-cell and cell-matrix adhesion. Overexpression of SUSD2 is observed in
- 62 -cancerous cells and SUSD2 is an established enrichment marker for various mesenchymal stem cells. Assaying D75 PRP 2.3 CBP for surface proteins revealed elevated SUSD2 expression on cells that labeled positive for recoverin, a neuronal-specific calcium-binding protein that is primarily expressed in photoreceptors. With this finding, SUSD2 was tested for use as an enrichment marker.
[00232]
SUSD2 had high co-expression with recoverin, a marker expressed by cells fated to become photoreceptors. Additionally, SUSD2 enriched for ¨95%
recoverin positive-positive cells compared to ¨83% recoverin-positive cell following CD171-enrichment (FIG. 31). Studies were performed on cells pre-treated with DAPT and plated on to LN521 with PD033 in the presence of DAPT. Recoverin coupled with CHX10, a transcription factor highly selective for early stage neural retinal progenitor cells, may be considered a hallmark characterization marker for (cone) bipolar cells (FIG. 34). Bipolar cells are late born retinal neurons which interconnect with photoreceptors and ganglion cells to facilitate signal transduction. CHX10-positive (recoverin-negative) cells may also be expressed in a subset of Muller glia cells.
[00233]
SUSD2 expression was investigated concurrent with RCVRN
expression for differentiation endpoints from D55 through D105, prior to MACS
enrichment (FIG. 32). The precision by which SUSD2 targets only PRP and no other retinal neurons was important for the final product's purity. Despite low cell yield following SUSD2 enrichment, the SUSD2 marker enriched recoverin-expressing cells at each time examined (Table 3) suggesting that SUSD2 is a highly specific marker for recoverin-positive possible PRP. FIG.
33 further demonstrates SUSD2-target specificity.
[00234] As previously discussed, CHX10 and recoverin are hallmark characterization markers for (cone) bipolar cells. CHX10 is expressed early in retinal progenitor cells and downregulates expression in post-mitotic PRP but remains highly expressed in post-mitotic bipolar cells and some Muller glia cells. The studies showed that CHX10 is low at the earlier (D65) timepoint, in both pre- and post-SUSD2 MACS-enrichment.
Additionally, recoverin-CHX10 expression is negligible at this time, suggesting there are no bipolar cells in the product following SUSD2-enrichment at this time.
[00235] Conversely, co-labeling recoverin and neuronal differentiation factor 1 (NeuroD1, ND1), a transiently expressed transcription factor that plays a role in the terminal
[00232]
SUSD2 had high co-expression with recoverin, a marker expressed by cells fated to become photoreceptors. Additionally, SUSD2 enriched for ¨95%
recoverin positive-positive cells compared to ¨83% recoverin-positive cell following CD171-enrichment (FIG. 31). Studies were performed on cells pre-treated with DAPT and plated on to LN521 with PD033 in the presence of DAPT. Recoverin coupled with CHX10, a transcription factor highly selective for early stage neural retinal progenitor cells, may be considered a hallmark characterization marker for (cone) bipolar cells (FIG. 34). Bipolar cells are late born retinal neurons which interconnect with photoreceptors and ganglion cells to facilitate signal transduction. CHX10-positive (recoverin-negative) cells may also be expressed in a subset of Muller glia cells.
[00233]
SUSD2 expression was investigated concurrent with RCVRN
expression for differentiation endpoints from D55 through D105, prior to MACS
enrichment (FIG. 32). The precision by which SUSD2 targets only PRP and no other retinal neurons was important for the final product's purity. Despite low cell yield following SUSD2 enrichment, the SUSD2 marker enriched recoverin-expressing cells at each time examined (Table 3) suggesting that SUSD2 is a highly specific marker for recoverin-positive possible PRP. FIG.
33 further demonstrates SUSD2-target specificity.
[00234] As previously discussed, CHX10 and recoverin are hallmark characterization markers for (cone) bipolar cells. CHX10 is expressed early in retinal progenitor cells and downregulates expression in post-mitotic PRP but remains highly expressed in post-mitotic bipolar cells and some Muller glia cells. The studies showed that CHX10 is low at the earlier (D65) timepoint, in both pre- and post-SUSD2 MACS-enrichment.
Additionally, recoverin-CHX10 expression is negligible at this time, suggesting there are no bipolar cells in the product following SUSD2-enrichment at this time.
[00235] Conversely, co-labeling recoverin and neuronal differentiation factor 1 (NeuroD1, ND1), a transiently expressed transcription factor that plays a role in the terminal
- 63 -differentiation of photoreceptors, showed high expression levels at D65 pre-and post-SUSD2 MACS-enrichment. Past D65, RCVRN-CHX10-expressing bipolar cells (and/or possibly some CHX10-expressing Muller glia cells) were observed. RCVRN-ND1-expression gradually downregulated and the combination was expressed in less than half of the enriched D85 cells compared to D65 and D75, likely consistent with the transient expression of ND1.
[00236]
Table 3: Performing MACS at several time points shows SUSD2 consistently improves the population's recoverin expression and yields high cell recovery at differentiation endpoint days 55-65. The high yield further illustrates high SUSD2 expression at these time points.
MACS
Recoverin% %Yield Pre- SUSD2 SUSD2 Batch Day MACS enrich enrich ADD PRP24 55 67.64% 81.37% 49.0%
ADD PRP25 55 65.70% 81.37% 43.6%
ADD PRP27 56 66.41% 73.29% 46.0%
ADD PRP28 59 80.45% 84.52% 39.74%
ADD PRP27 65 88.21% 91.56% 29.21%
ADD PRP24 65 78.97% 92.51% 34.9%
MJS PRP28 75 44.37% 79.33% 7.4%
ADD PRP24 76 72.38% 88.05% 3.5%
ADD PRP23 78 41.99% 75.16% 2.6%
[00237]
These studies, coupled with peak SUSD2 expression time, suggested that SUSD2 is a good enrichment marker at early time points, such as D65.
Furthermore, at D65, SUSD2 was highly selective for RCVRN- and ND1-expressing cells but not for CHX10-expressing (off-target or progenitor) cells. The selective attribute of SUSD2 was highlighted by the enrichment of a highly recoverin-positive pure population even when cell yield post-enrichment was marginal. Overall, SUSD2 is a valuable target marker for PRP
enrichment provided the final product is manufactured at early time points when SUSD2 expression is high, such as D55 and D65.
[00238] In addition to SUSD2 and CD171, alternative potential PRP enrichment markers were pinpointed. These surface markers also co-localized with recoverin, a neuronal-specific calcium-binding protein that is primarily expressed in photoreceptors and cone bipolar cells (Gunhan et al., 2003; Haverkamp et al., 2003), although to a lesser extent. Table 4 lists 15 additional potential PRP enrichment markers, excluding CD171 and SUSD2.
[00236]
Table 3: Performing MACS at several time points shows SUSD2 consistently improves the population's recoverin expression and yields high cell recovery at differentiation endpoint days 55-65. The high yield further illustrates high SUSD2 expression at these time points.
MACS
Recoverin% %Yield Pre- SUSD2 SUSD2 Batch Day MACS enrich enrich ADD PRP24 55 67.64% 81.37% 49.0%
ADD PRP25 55 65.70% 81.37% 43.6%
ADD PRP27 56 66.41% 73.29% 46.0%
ADD PRP28 59 80.45% 84.52% 39.74%
ADD PRP27 65 88.21% 91.56% 29.21%
ADD PRP24 65 78.97% 92.51% 34.9%
MJS PRP28 75 44.37% 79.33% 7.4%
ADD PRP24 76 72.38% 88.05% 3.5%
ADD PRP23 78 41.99% 75.16% 2.6%
[00237]
These studies, coupled with peak SUSD2 expression time, suggested that SUSD2 is a good enrichment marker at early time points, such as D65.
Furthermore, at D65, SUSD2 was highly selective for RCVRN- and ND1-expressing cells but not for CHX10-expressing (off-target or progenitor) cells. The selective attribute of SUSD2 was highlighted by the enrichment of a highly recoverin-positive pure population even when cell yield post-enrichment was marginal. Overall, SUSD2 is a valuable target marker for PRP
enrichment provided the final product is manufactured at early time points when SUSD2 expression is high, such as D55 and D65.
[00238] In addition to SUSD2 and CD171, alternative potential PRP enrichment markers were pinpointed. These surface markers also co-localized with recoverin, a neuronal-specific calcium-binding protein that is primarily expressed in photoreceptors and cone bipolar cells (Gunhan et al., 2003; Haverkamp et al., 2003), although to a lesser extent. Table 4 lists 15 additional potential PRP enrichment markers, excluding CD171 and SUSD2.
- 64 -[00239] Table 4: Surface proteins evaluated for PRP enrichment.
%pmti.gfefifftmvesss$
33eq.qm (e..13 411esi=rsr> mow:We tv.ifitatio,s z.Ketzite C:Da (M) 37.5 U.4 rAttr Qcvfe pregpx,serk i:e4le ms the v3smisyymn M52 LAM-3) hiPSC-cik<ed cam ishatocmistm nwtam tnt~. 35.3.
lermisptas prnteitl3ncaiizig aroi tostSatentE
36.7 P3..1 plasm rrombram, ckenaie5 rost= mit** aPE
31t P3.3. nfinealki WNSertiskntekvussfa: P3.3. 3.6 ("Din Wohe.A(..1333 Retkof clitvelopmeht C:133 Won* niC31 OvetwaleM
M3.47 fuettim attilske. 4:4.5 Chtmc=Miv re<ti.ltor tha fz3ONta cataa c3h,..axx&-tic M3.84 'f.X7t.43 33.8 53-3 vity =
3"13.1Ø3 maintehame tetitia$ PoRmtaiis 3,5S3 63.2 02:30 mote4 35.S 1.5.a t'cauPtr ;.=etivatitx{ P3.7 58.7 C332X4 5txthanjotawshgn-trampi.Ist.'m .ktPeett het:3,3 36.7 Ex3,5 1:3)3440.?1.n3tx1 .4,soW=ix.^.1<kvelopme=rsi.. 4:11.,e3twomrtt 33.3 KA. KAM FiAxt0415 pratM spia fthr-insiiw*d Tetino3 ilege,:wotIon '14,1 Fig7 (Plit xfaxesim. siva3mtlitittaivs5, &PC 13.6 35:2 [00240]
Investigations into these surface molecules were primarily focused on cellular expression and/or function within the developing or adult retina.
Table 4 lists the percent of the cell population that either co-expressed the surface antigen with recoverin or expressed the surface antigen exclusively on off-target differentiated cells, respectively.
Percent population expressing either of both markers was determined by flow cytometric analysis of dual-stained D75 PRP product (FIG. 15). Additionally, percent population of cells expressing only recoverin (in FITC, x-axis) or only the surface antigen (in APC, y-axis) was evaluated. All plots were gated against unstained (empty) cells and the corresponding isotype control (REA IgGl, MsIgGl, Ms IgG2a, MsIgG2b, MsIgM). The surface molecules with high recoverin co-expression yet low off-target expression (<20%) include CD11, CD133, CD230, and CD344.
%pmti.gfefifftmvesss$
33eq.qm (e..13 411esi=rsr> mow:We tv.ifitatio,s z.Ketzite C:Da (M) 37.5 U.4 rAttr Qcvfe pregpx,serk i:e4le ms the v3smisyymn M52 LAM-3) hiPSC-cik<ed cam ishatocmistm nwtam tnt~. 35.3.
lermisptas prnteitl3ncaiizig aroi tostSatentE
36.7 P3..1 plasm rrombram, ckenaie5 rost= mit** aPE
31t P3.3. nfinealki WNSertiskntekvussfa: P3.3. 3.6 ("Din Wohe.A(..1333 Retkof clitvelopmeht C:133 Won* niC31 OvetwaleM
M3.47 fuettim attilske. 4:4.5 Chtmc=Miv re<ti.ltor tha fz3ONta cataa c3h,..axx&-tic M3.84 'f.X7t.43 33.8 53-3 vity =
3"13.1Ø3 maintehame tetitia$ PoRmtaiis 3,5S3 63.2 02:30 mote4 35.S 1.5.a t'cauPtr ;.=etivatitx{ P3.7 58.7 C332X4 5txthanjotawshgn-trampi.Ist.'m .ktPeett het:3,3 36.7 Ex3,5 1:3)3440.?1.n3tx1 .4,soW=ix.^.1<kvelopme=rsi.. 4:11.,e3twomrtt 33.3 KA. KAM FiAxt0415 pratM spia fthr-insiiw*d Tetino3 ilege,:wotIon '14,1 Fig7 (Plit xfaxesim. siva3mtlitittaivs5, &PC 13.6 35:2 [00240]
Investigations into these surface molecules were primarily focused on cellular expression and/or function within the developing or adult retina.
Table 4 lists the percent of the cell population that either co-expressed the surface antigen with recoverin or expressed the surface antigen exclusively on off-target differentiated cells, respectively.
Percent population expressing either of both markers was determined by flow cytometric analysis of dual-stained D75 PRP product (FIG. 15). Additionally, percent population of cells expressing only recoverin (in FITC, x-axis) or only the surface antigen (in APC, y-axis) was evaluated. All plots were gated against unstained (empty) cells and the corresponding isotype control (REA IgGl, MsIgGl, Ms IgG2a, MsIgG2b, MsIgM). The surface molecules with high recoverin co-expression yet low off-target expression (<20%) include CD11, CD133, CD230, and CD344.
- 65 -[00241]
This type of flow cytometric profile increases the likelihood of the antigen being expressed on a greater number of PRP cells and fewer off-target cells. To determine if this was indeed the case, differentiated cells from multiple experiments were enriched for PRP using antibodies against CD111, CD230, CD344 and CD133 surface antigens. FIG. 16 shows recoverin expression at D55, D65 and D75. Recoverin expression peaked at D65 for all conditions, including pre-MACS (CD133-enrichment was only performed on D75 PRP, single time point, diamond) and dropped by D75. The data suggests that D65 may be the optimal time point for PRP enrichment with these specific surface antigens (excluding CD133).
[00242] FIGS. 17-22 display a tabular and graphical representation of the percent expression of recoverin (on-target PRP marker) with off-target cell markers Pax6 (expressed by retinal progenitor cells-RPC, amacrine cells-AC and retinal ganglion cells-RGC), Onecutl (expressed by RPCs and horizontal cells), Ki67 (expressed by proliferating cells) and CHX10 (expressed by RPC and co-expressed with recoverin on bipolar cells). The highest recoverin expression occurred with CD344-enrichment. The eluted fraction for each enrichment showed some recoverin-positive cells but overall a lower fraction than the enriched portion while showing a higher percentage of the off-target marker Onecutl. There were essentially no proliferating cells or CHX10+ RPC/bipolar off-target cells in the cell populations evaluated.
Again, the CD344-enriched cells showed the greatest percentage of recoverin-positive expression and low Pax6 and Onecutl. There were no proliferating cells in these cell populations.
[00243]
There was found to be an overall reduction of on-target and off-target cells by D75, suggesting that the expression of the target markers may have significantly reduced by this time point. Interestingly, CHX10 expression was high, suggestive of possible bipolar off-target cells and/or a possible second wave of progenitor cells.
[00244] For all studies, recoverin expression was the greatest post-CD344-enrichment followed by post-CD230-enrichment. CD344 enrichment also reduced Pax6-positive and CHX10-positive off-target cell markers compared to pre-MACS. The elute or flow-through for all three enrichments appeared to contain majority of Pax6 +
and CHX10+
cells, suggesting that the enrichment was targeting recoverin + PRPs and not off-target cells such as retinal ganglion cells (RGCs) or amacrine cells (ACs), both of which express Pax6.
Since the enriched portion expressed moderate levels of CHX10 at day 75, some portion of the
This type of flow cytometric profile increases the likelihood of the antigen being expressed on a greater number of PRP cells and fewer off-target cells. To determine if this was indeed the case, differentiated cells from multiple experiments were enriched for PRP using antibodies against CD111, CD230, CD344 and CD133 surface antigens. FIG. 16 shows recoverin expression at D55, D65 and D75. Recoverin expression peaked at D65 for all conditions, including pre-MACS (CD133-enrichment was only performed on D75 PRP, single time point, diamond) and dropped by D75. The data suggests that D65 may be the optimal time point for PRP enrichment with these specific surface antigens (excluding CD133).
[00242] FIGS. 17-22 display a tabular and graphical representation of the percent expression of recoverin (on-target PRP marker) with off-target cell markers Pax6 (expressed by retinal progenitor cells-RPC, amacrine cells-AC and retinal ganglion cells-RGC), Onecutl (expressed by RPCs and horizontal cells), Ki67 (expressed by proliferating cells) and CHX10 (expressed by RPC and co-expressed with recoverin on bipolar cells). The highest recoverin expression occurred with CD344-enrichment. The eluted fraction for each enrichment showed some recoverin-positive cells but overall a lower fraction than the enriched portion while showing a higher percentage of the off-target marker Onecutl. There were essentially no proliferating cells or CHX10+ RPC/bipolar off-target cells in the cell populations evaluated.
Again, the CD344-enriched cells showed the greatest percentage of recoverin-positive expression and low Pax6 and Onecutl. There were no proliferating cells in these cell populations.
[00243]
There was found to be an overall reduction of on-target and off-target cells by D75, suggesting that the expression of the target markers may have significantly reduced by this time point. Interestingly, CHX10 expression was high, suggestive of possible bipolar off-target cells and/or a possible second wave of progenitor cells.
[00244] For all studies, recoverin expression was the greatest post-CD344-enrichment followed by post-CD230-enrichment. CD344 enrichment also reduced Pax6-positive and CHX10-positive off-target cell markers compared to pre-MACS. The elute or flow-through for all three enrichments appeared to contain majority of Pax6 +
and CHX10+
cells, suggesting that the enrichment was targeting recoverin + PRPs and not off-target cells such as retinal ganglion cells (RGCs) or amacrine cells (ACs), both of which express Pax6.
Since the enriched portion expressed moderate levels of CHX10 at day 75, some portion of the
- 66 -recoverin-labeled cells may also be co-localized on CHX10-labeled cells, suggesting there was a population of bipolar cells (BPs) in the enriched fraction.
[00245]
Enrichment using CD133 was done on a different cell line (31538.102) to test whether the line differentiated towards PRP. CD133 enriched for recoverin+ cells and there was an overall reduction in off-target cell marker expression post-enrichment compared to pre-MACS. While Ki67 also reduced post-enrichment, PD0332991 treatment removed any remaining proliferating cells present in the enriched fraction.
[00246]
Thus, the markers CD71, SUSD2, CD111, CD133, CD230, and CD344 may be used for the enrichment of the PRP cell populations and removal of off-target cells.
Example 8¨ PRP Enrichment using CliniMACSO
[00247]
Further studies were conducted to assess the feasibility of PRP
enrichment on a higher throughput scale, such as using the CliniMACSO
instrument. PRP
enrichment using the CD171 marker was tested using the CliniMACS instrument whereby the C-1 or C-2 cells were input to the instrument using the CD34.2 or Enrichment 1.1 program with LS tubing. The PRP-enriched cells were then assessed for TUBB3/Nestin and RCVRN.
The CliniMACS high-throughput method had similar output purity as compared to the LS
Column method with more than 90% of the output cells being neurons and more than 50% of the cells being positive for RCVRN (FIGS. 3 and 9).
Example 9¨ Materials and Methods [00248] The flow cytometry wash buffer was prepared by adding 20 mL FBS or human serum albumin to 1000 mL of DPBS (i.e., without calcium and magnesium).
The buffer was filter sterilized and can be stored at 4 C for up to 4 weeks.
[00249] The flow cytometry permeabilization buffer was prepared by adding 20 mL FBS to 1000 mL DPBS (i.e., without calcium and magnesium). One gram of Saponin was added and mixed well. The buffer was filter sterilized and can be stored at 4 C for up to 4 weeks.
[00250] The flow cytometry Live-Dead Red stain was prepared by diluting Live-Dead Stain 1:1000 in DPBS (i.e., without calcium and magnesium). One mL of the stain was prepared per 2x106 cells being assayed. The stain was prepared fresh before use.
[00245]
Enrichment using CD133 was done on a different cell line (31538.102) to test whether the line differentiated towards PRP. CD133 enriched for recoverin+ cells and there was an overall reduction in off-target cell marker expression post-enrichment compared to pre-MACS. While Ki67 also reduced post-enrichment, PD0332991 treatment removed any remaining proliferating cells present in the enriched fraction.
[00246]
Thus, the markers CD71, SUSD2, CD111, CD133, CD230, and CD344 may be used for the enrichment of the PRP cell populations and removal of off-target cells.
Example 8¨ PRP Enrichment using CliniMACSO
[00247]
Further studies were conducted to assess the feasibility of PRP
enrichment on a higher throughput scale, such as using the CliniMACSO
instrument. PRP
enrichment using the CD171 marker was tested using the CliniMACS instrument whereby the C-1 or C-2 cells were input to the instrument using the CD34.2 or Enrichment 1.1 program with LS tubing. The PRP-enriched cells were then assessed for TUBB3/Nestin and RCVRN.
The CliniMACS high-throughput method had similar output purity as compared to the LS
Column method with more than 90% of the output cells being neurons and more than 50% of the cells being positive for RCVRN (FIGS. 3 and 9).
Example 9¨ Materials and Methods [00248] The flow cytometry wash buffer was prepared by adding 20 mL FBS or human serum albumin to 1000 mL of DPBS (i.e., without calcium and magnesium).
The buffer was filter sterilized and can be stored at 4 C for up to 4 weeks.
[00249] The flow cytometry permeabilization buffer was prepared by adding 20 mL FBS to 1000 mL DPBS (i.e., without calcium and magnesium). One gram of Saponin was added and mixed well. The buffer was filter sterilized and can be stored at 4 C for up to 4 weeks.
[00250] The flow cytometry Live-Dead Red stain was prepared by diluting Live-Dead Stain 1:1000 in DPBS (i.e., without calcium and magnesium). One mL of the stain was prepared per 2x106 cells being assayed. The stain was prepared fresh before use.
- 67 -[00251] The flow cytometry fixation buffer was prepared by adding 110 pi of 36.5% Formaldehyde to 880 pi of DPBS (i.e. without calcium and magnesium). One mL of stain was prepared per 2x106 cells being assayed. The buffer was prepared fresh before use.
Example 10 ¨ Depletion Markers for PRP Enrichment [00252] Additionally, surface molecules were identified which could be used as depletion markers to inversely enrich the PRP cell population. These molecules did not show co-expression with recoverin but did label off-target cells, making them good depletion markers for unwanted cell types. Table 5 lists four possible depletion candidates that showed very little co-expression with recoverin (<15%) but labeled other cell types (>30%). Expression profiles of recoverin against the depletion antibodies were evaluated by flow analysis. FIG. 21 shows the low expression or near absence of depletion candidate surface antigen co-expression with recoverin.
[00253] Table 5: Depletion markers for PRP enrichment.
% co-expression with %
antigen Antigen Description RCVRN only CD9 Integral membrane protein associated with integrins 5.4 32.7 CD49f Integrin alpha 6 11 41.2 CD340 Cell surface receptor tyrosine protein kinase ErbB2 3.6 31.5 Podoplanin Mucin-type transmembrane protein 3.6 32.5 Example 11 ¨ Optimization of PRP Differentiation [00254] The PRP differentiation method was further evaluated for use of the Wnt activator, such as CKI-7, at Days 0 and 1. The cells were differentiated in the absence of CM-7 and characterized for PAX6, CHX10, Ki67, and PMEL expression at Day 15 and Day 25 of the differentiation process. It was found that without CM-7 at Days 0 and 1, the cell population had a similar expression of PAX6, CHX10, Ki67, and PMEL at Days 15 and 25 (FIGS. 22-25).
Thus, the PRP cells may be differentiated in the absence of CM-7 at Days 0 and 1.
Example 10 ¨ Depletion Markers for PRP Enrichment [00252] Additionally, surface molecules were identified which could be used as depletion markers to inversely enrich the PRP cell population. These molecules did not show co-expression with recoverin but did label off-target cells, making them good depletion markers for unwanted cell types. Table 5 lists four possible depletion candidates that showed very little co-expression with recoverin (<15%) but labeled other cell types (>30%). Expression profiles of recoverin against the depletion antibodies were evaluated by flow analysis. FIG. 21 shows the low expression or near absence of depletion candidate surface antigen co-expression with recoverin.
[00253] Table 5: Depletion markers for PRP enrichment.
% co-expression with %
antigen Antigen Description RCVRN only CD9 Integral membrane protein associated with integrins 5.4 32.7 CD49f Integrin alpha 6 11 41.2 CD340 Cell surface receptor tyrosine protein kinase ErbB2 3.6 31.5 Podoplanin Mucin-type transmembrane protein 3.6 32.5 Example 11 ¨ Optimization of PRP Differentiation [00254] The PRP differentiation method was further evaluated for use of the Wnt activator, such as CKI-7, at Days 0 and 1. The cells were differentiated in the absence of CM-7 and characterized for PAX6, CHX10, Ki67, and PMEL expression at Day 15 and Day 25 of the differentiation process. It was found that without CM-7 at Days 0 and 1, the cell population had a similar expression of PAX6, CHX10, Ki67, and PMEL at Days 15 and 25 (FIGS. 22-25).
Thus, the PRP cells may be differentiated in the absence of CM-7 at Days 0 and 1.
- 68 -[00255]
Alternatively, the differentiation method was performed without the use of RIM. Instead, the cells were cultured in the RD1 media supplemented with LDN193189 for Day 0-Day 1 and then cultured in RD2 media without LDN193189 (FIGS. 26-30).
[00256] The method of Examples 1-3 was modified to remove the two-day culture in RIM media and the early neural retinal differentiation was evaluated. The cells were directly cultured in the RD1 media with the high concentrations of LDN193189, SB431542, and CKI-7 for Day 0 to Day 1 followed by culture in the RD2 media without LDN193189 Day 2 to Day 10. The cells were aggregated at Day 15 and samples were processed for flow analysis at time of aggregation, at Day 30 mid-process development and Day 75 end-process development.
[00257] The D15 in-process flow analysis showed that although expression of PAX6, an early human neuroectoderm cell fate determinant, was similar in CBP
2.3 (with RIM) vs CBP 2.5 (without RIM) conditions, expression of early eye field marker CHX10 was low in RIM conditions (FIG. 26). Removal of the RIM step with RD1 with LDN was observed to increase CHX10 expression. Similarly, removal of RIM reduced expression of the RPE marker Tyrpl by nearly 50% compared to conditions containing RIM (FIGS. 27 and 28).
[00258]
Multiple cell lines were tested to determine if removal of the RIM
promoted PRP differentiation across the various lines. Lines 31536.102, 31538.101 and 31538.102 were lines that consistently expressed low CHX10 at D15 in-process analysis but CHX10 expression in both lines significantly increased when the RIM step was removed (FIG.
27). FIG. 27 compares expression of neuroectoderm marker Pax6, eye field marker CHX10, and RPE markers Tyrpl and PMEL across 4 lines in the presence of RIM (CBP) or in its absence (-RIM). Across lines, Pax expression was similar in the presence or absence of RIM.
CHX10 expression increased across lines when RIM was removed but dramatically improved in lines 8.101 and 8.102, by about 80% in both lines. Co-incidentally, Tyrpl expression was reduced following removal of RIM.
[00259]
Pigmented-cell-specific protein (PMEL) is generally associated with RPE pigmentation. In the present differentiation system, it was shown that PMEL expression remains high at D15 and low PMEL expression at D15 usually results in poor PRP
differentiation. Removal of RIM does not disrupt PMEL expression in either line 6.102 or line A but markedly increases PMEL expression in lines 8.101 and 8.102. This demonstrated that
Alternatively, the differentiation method was performed without the use of RIM. Instead, the cells were cultured in the RD1 media supplemented with LDN193189 for Day 0-Day 1 and then cultured in RD2 media without LDN193189 (FIGS. 26-30).
[00256] The method of Examples 1-3 was modified to remove the two-day culture in RIM media and the early neural retinal differentiation was evaluated. The cells were directly cultured in the RD1 media with the high concentrations of LDN193189, SB431542, and CKI-7 for Day 0 to Day 1 followed by culture in the RD2 media without LDN193189 Day 2 to Day 10. The cells were aggregated at Day 15 and samples were processed for flow analysis at time of aggregation, at Day 30 mid-process development and Day 75 end-process development.
[00257] The D15 in-process flow analysis showed that although expression of PAX6, an early human neuroectoderm cell fate determinant, was similar in CBP
2.3 (with RIM) vs CBP 2.5 (without RIM) conditions, expression of early eye field marker CHX10 was low in RIM conditions (FIG. 26). Removal of the RIM step with RD1 with LDN was observed to increase CHX10 expression. Similarly, removal of RIM reduced expression of the RPE marker Tyrpl by nearly 50% compared to conditions containing RIM (FIGS. 27 and 28).
[00258]
Multiple cell lines were tested to determine if removal of the RIM
promoted PRP differentiation across the various lines. Lines 31536.102, 31538.101 and 31538.102 were lines that consistently expressed low CHX10 at D15 in-process analysis but CHX10 expression in both lines significantly increased when the RIM step was removed (FIG.
27). FIG. 27 compares expression of neuroectoderm marker Pax6, eye field marker CHX10, and RPE markers Tyrpl and PMEL across 4 lines in the presence of RIM (CBP) or in its absence (-RIM). Across lines, Pax expression was similar in the presence or absence of RIM.
CHX10 expression increased across lines when RIM was removed but dramatically improved in lines 8.101 and 8.102, by about 80% in both lines. Co-incidentally, Tyrpl expression was reduced following removal of RIM.
[00259]
Pigmented-cell-specific protein (PMEL) is generally associated with RPE pigmentation. In the present differentiation system, it was shown that PMEL expression remains high at D15 and low PMEL expression at D15 usually results in poor PRP
differentiation. Removal of RIM does not disrupt PMEL expression in either line 6.102 or line A but markedly increases PMEL expression in lines 8.101 and 8.102. This demonstrated that
- 69 -removal of RIM does not hinder the PRP developmental process but benefits lines which require an extra push towards PRP development. Taken together, the data suggests that removal of RIM may reduce the potential for D15 cells to differentiate into RPE while increasing the possibility of differentiating into PRP.
[00260] Based on historical data, during mid-stage (in-process) PRP
development (¨D30), expression of PAX6 and CHX10 peaks while TYRP1 and PMEL
expression further reduces. While the developmental process naturally drives these changes in protein expression, the removal of RIM at the early stages of differentiation provides additional support in lines previously considered unacceptable for PRP differentiation.
Early removal of RIM affects D30 mid-stage PRP development in lines 8.101 and 8.102 by increasing PAX6 and CHX10 expression levels and reducing TRYP1 and PMEL expression levels.
[00261] As the retina develops, a heterogenous population of retinal cells are born in a chronological sequence and characterization of these cell populations are based on a specific panel of antibodies targeted to antigens expressed by the off-target cell types. Current panels to identify on-target PRP cells include recoverin (RCVRN) coupled with either NeuroD1 (neuronal differentiation factor 1, ND1; FIG. 29A) or with CHX10 (FIG.
29B).
Recoverin is a neuronal-specific calcium-binding protein that is primarily expressed in photoreceptors and NeuroD1 is a transiently expressed transcription factor that plays a role in the terminal differentiation of photoreceptors. CHX10 coupled with RCVRN is a hallmark characterization marker for (cone) bipolar cells. While CHX10 is expressed early in retinal progenitor cells, it downregulates expression in post-mitotic PRP but remains highly expressed in post-mitotic bipolar cells and some Muller glia cells. Bipolar cells will express both CHX10 and RCVRN while PRP will be only RCVRN+ (positive) and CHX10- (negative).
Hence, the dual labeling with RCVRN and CHX10 was used to validate the absence or low presence of bipolar cells in the cell population and demonstrate that RCVRN+ cells are primarily differentiated PRP.
[00262] The effect of no RIM on D75 PRP was most evident in Line A, whereby RCVRN and ND1 expression increased with the removal of RIM. This data showed that removal of RIM impacted PRP development. Furthermore, CHX10 expression decreased or remained <10% across lines with the removal of RIM, resulting in a reduction of RCVRN+/CHX10+ double-positive cells as well.
[00260] Based on historical data, during mid-stage (in-process) PRP
development (¨D30), expression of PAX6 and CHX10 peaks while TYRP1 and PMEL
expression further reduces. While the developmental process naturally drives these changes in protein expression, the removal of RIM at the early stages of differentiation provides additional support in lines previously considered unacceptable for PRP differentiation.
Early removal of RIM affects D30 mid-stage PRP development in lines 8.101 and 8.102 by increasing PAX6 and CHX10 expression levels and reducing TRYP1 and PMEL expression levels.
[00261] As the retina develops, a heterogenous population of retinal cells are born in a chronological sequence and characterization of these cell populations are based on a specific panel of antibodies targeted to antigens expressed by the off-target cell types. Current panels to identify on-target PRP cells include recoverin (RCVRN) coupled with either NeuroD1 (neuronal differentiation factor 1, ND1; FIG. 29A) or with CHX10 (FIG.
29B).
Recoverin is a neuronal-specific calcium-binding protein that is primarily expressed in photoreceptors and NeuroD1 is a transiently expressed transcription factor that plays a role in the terminal differentiation of photoreceptors. CHX10 coupled with RCVRN is a hallmark characterization marker for (cone) bipolar cells. While CHX10 is expressed early in retinal progenitor cells, it downregulates expression in post-mitotic PRP but remains highly expressed in post-mitotic bipolar cells and some Muller glia cells. Bipolar cells will express both CHX10 and RCVRN while PRP will be only RCVRN+ (positive) and CHX10- (negative).
Hence, the dual labeling with RCVRN and CHX10 was used to validate the absence or low presence of bipolar cells in the cell population and demonstrate that RCVRN+ cells are primarily differentiated PRP.
[00262] The effect of no RIM on D75 PRP was most evident in Line A, whereby RCVRN and ND1 expression increased with the removal of RIM. This data showed that removal of RIM impacted PRP development. Furthermore, CHX10 expression decreased or remained <10% across lines with the removal of RIM, resulting in a reduction of RCVRN+/CHX10+ double-positive cells as well.
- 70 -[00263] Off-target cell markers include Pax6-Isl 1 double-positive for retinal ganglion cells (RGC), HNF6 for horizontal cells and Ki67, a pan proliferative cell marker. Pax6 is also expressed on other mature retinal cells, as described below (FIG. 30).
Pax6 is a reliable marker for neural induction but in the retina, several post-mitotic off-target cells express Pax6, such as RGCs, Muller glia and a subset of amacrine cells. Likewise, Is11 is also expressed by multiple mature off-target retinal cells, such as ON bipolar cells, RGCs and a subset of amacrine cells. At this developmental stage, the no RIM condition did not make much of an impact other than a slight reduction in off-target marker expression. The double positive Isl1/Pax6 population represented a small fraction (<10%) of lingering RGCs or amacrine cell subset, regardless of the media condition. Additionally, there were negligible HNF6+ horizontal cells and virtually no proliferating cells across lines and conditions.
[00264]
Thus, the present studies showed that removing RIM from the media sequences used for differentiation significantly impacts PRP differentiation and development, especially in the early developmental stages. The presence of RIM attenuated expression at D15 across all lines at in some lines at D30. Removing RIM
appeared to "rescue"
the reduced CHX10 expression, across lines at D15 and at D30 a significant improvement was observed in lines 8.101 and 8.102. Most importantly, removing RIM increased RCVRN and ND1 expression levels and reduced CHX10 levels in some cases but did not have a disadvantageous effect at any time point observed.
****
[00265] All methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Pax6 is a reliable marker for neural induction but in the retina, several post-mitotic off-target cells express Pax6, such as RGCs, Muller glia and a subset of amacrine cells. Likewise, Is11 is also expressed by multiple mature off-target retinal cells, such as ON bipolar cells, RGCs and a subset of amacrine cells. At this developmental stage, the no RIM condition did not make much of an impact other than a slight reduction in off-target marker expression. The double positive Isl1/Pax6 population represented a small fraction (<10%) of lingering RGCs or amacrine cell subset, regardless of the media condition. Additionally, there were negligible HNF6+ horizontal cells and virtually no proliferating cells across lines and conditions.
[00264]
Thus, the present studies showed that removing RIM from the media sequences used for differentiation significantly impacts PRP differentiation and development, especially in the early developmental stages. The presence of RIM attenuated expression at D15 across all lines at in some lines at D30. Removing RIM
appeared to "rescue"
the reduced CHX10 expression, across lines at D15 and at D30 a significant improvement was observed in lines 8.101 and 8.102. Most importantly, removing RIM increased RCVRN and ND1 expression levels and reduced CHX10 levels in some cases but did not have a disadvantageous effect at any time point observed.
****
[00265] All methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
- 71 -REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Amit etal., Dev. Bio., 227:271-278, 2000.
Buchholz etal., Stem Cells, 27: 2427-2434, 2009.
Byrne etal., Nature, 450(7169):497-502, 2007.
Comyn etal., Curr. Opin. Neurol. 1, 4-9, 2010.
Gunhan etal., J Neurosci. 23(4)1383-1389, 2003.
Haverkamp etal., J Comp Neurol. 455(4):463-476, 2003.
Hirami etal., Neurosci. Lett., 48: 126-131, 2009.
Kanemura etal., PLoS One, 9, 2014.
Ludwig etal., Nat. Biotechnol., 24:185-187, 2006b.
Ludwig etal., Nat. Methods, 3:637-646, 2006a.
Meyer etal., PNAS, 106(39): 16698-16703, 2009.
PCT Publication No. WO 2007/069666 Al.
PCT Publication No. WO 2014/121077.
Pearson etal., Nature 485, 99-103, 2012.
Smith, In: Origins and Properties of Mouse Embryonic Stem Cells, 2000.
Strauss etal., Physiological Reviews, 85:845-881, 2005.
Takahashi etal., Cell, 126, 663-676, 2006.
Takahashi etal., Cell, 131, 861-872, 2007.
Thomson and Marshall, Curr. Top. Dev. Biol., 38:133-165, 1998.
Thomson and Odorico, Trends Biotechnol., 18(2):53-57, 2000.
Thomson etal. Proc. Natl. Acad. Scie. USA, 92:7844-7848, 1995.
U.S. Patent Application No. 2002/0076747.
U.S. Patent Application No. 2009/0246875.
U.S. Patent Application No. 2010/0210014.
U.S. Patent Application No. 2012/0196360.
U.S. Patent Application No. 2012/0276636.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Amit etal., Dev. Bio., 227:271-278, 2000.
Buchholz etal., Stem Cells, 27: 2427-2434, 2009.
Byrne etal., Nature, 450(7169):497-502, 2007.
Comyn etal., Curr. Opin. Neurol. 1, 4-9, 2010.
Gunhan etal., J Neurosci. 23(4)1383-1389, 2003.
Haverkamp etal., J Comp Neurol. 455(4):463-476, 2003.
Hirami etal., Neurosci. Lett., 48: 126-131, 2009.
Kanemura etal., PLoS One, 9, 2014.
Ludwig etal., Nat. Biotechnol., 24:185-187, 2006b.
Ludwig etal., Nat. Methods, 3:637-646, 2006a.
Meyer etal., PNAS, 106(39): 16698-16703, 2009.
PCT Publication No. WO 2007/069666 Al.
PCT Publication No. WO 2014/121077.
Pearson etal., Nature 485, 99-103, 2012.
Smith, In: Origins and Properties of Mouse Embryonic Stem Cells, 2000.
Strauss etal., Physiological Reviews, 85:845-881, 2005.
Takahashi etal., Cell, 126, 663-676, 2006.
Takahashi etal., Cell, 131, 861-872, 2007.
Thomson and Marshall, Curr. Top. Dev. Biol., 38:133-165, 1998.
Thomson and Odorico, Trends Biotechnol., 18(2):53-57, 2000.
Thomson etal. Proc. Natl. Acad. Scie. USA, 92:7844-7848, 1995.
U.S. Patent Application No. 2002/0076747.
U.S. Patent Application No. 2009/0246875.
U.S. Patent Application No. 2010/0210014.
U.S. Patent Application No. 2012/0196360.
U.S. Patent Application No. 2012/0276636.
- 72 -U.S. Patent No. 5,843,780.
U.S. Patent No. 6,103,470.
U.S. Patent No. 6,200,806.
U.S. Patent No. 6,416,998.
U.S. Patent No. 6,833,269.
U.S. Patent No. 7,029,913.
U.S. Patent No. 7,442,548.
U.S. Patent No. 7,598,364.
U.S. Patent No. 7,682,828.
U.S. Patent No. 7,989,425.
U.S. Patent No. 8,058,065.
U.S. Patent No. 8,071,369.
U.S. Patent No. 8,129,187.
U.S. Patent No. 8,268,620.
U.S. Patent No. 8,278,620.
U.S. Patent No. 8,546,140.
U.S. Patent No. 8,546,140.
U.S. Patent No. 8,741,648.
U.S. Patent Publication No. 2003/0211603.
U.S. Patent Publication No. 2010/0003757.
Xu etal., Nat. Biotechnol., 19:971-974, 2001.
Ying etal., Cell, 115:281-292, 2003.
Yu et al., Science, 318: 1917-1920, 2007.
Zhong et., Nature Communications, 5:4047, 2014.
U.S. Patent No. 6,103,470.
U.S. Patent No. 6,200,806.
U.S. Patent No. 6,416,998.
U.S. Patent No. 6,833,269.
U.S. Patent No. 7,029,913.
U.S. Patent No. 7,442,548.
U.S. Patent No. 7,598,364.
U.S. Patent No. 7,682,828.
U.S. Patent No. 7,989,425.
U.S. Patent No. 8,058,065.
U.S. Patent No. 8,071,369.
U.S. Patent No. 8,129,187.
U.S. Patent No. 8,268,620.
U.S. Patent No. 8,278,620.
U.S. Patent No. 8,546,140.
U.S. Patent No. 8,546,140.
U.S. Patent No. 8,741,648.
U.S. Patent Publication No. 2003/0211603.
U.S. Patent Publication No. 2010/0003757.
Xu etal., Nat. Biotechnol., 19:971-974, 2001.
Ying etal., Cell, 115:281-292, 2003.
Yu et al., Science, 318: 1917-1920, 2007.
Zhong et., Nature Communications, 5:4047, 2014.
- 73 -
Claims (108)
1. A method for producing a population of neural retinal progenitors (NRPs) comprising:
(a) obtaining a starting population of human induced pluripotent stem cells (iPSCs);
(b) culturing the cells in a first retinal differentiation medium (RD1) comprising a BMP inhibitor to further differentiate the cells to anterior neuroectoderm cells;
(c) inducing retinal differentiation of the anterior neuroectoderm cells by culturing the cells in a second retinal differentiation medium (RD2) essentially free of BMP
inhibitors to form retinal progenitor cells (RPCs); and (d) culturing the RPCs in a retinal maturation (RM) medium to produce NRPs.
(a) obtaining a starting population of human induced pluripotent stem cells (iPSCs);
(b) culturing the cells in a first retinal differentiation medium (RD1) comprising a BMP inhibitor to further differentiate the cells to anterior neuroectoderm cells;
(c) inducing retinal differentiation of the anterior neuroectoderm cells by culturing the cells in a second retinal differentiation medium (RD2) essentially free of BMP
inhibitors to form retinal progenitor cells (RPCs); and (d) culturing the RPCs in a retinal maturation (RM) medium to produce NRPs.
2. The method of claim 1, further comprising culturing the iPSCs in retinal induction medium (RIM) to initiate differentiation of the cells into anterior neuroectoderm cells prior to culturing the cells in RD1.
3. The method of claim 1, further comprising culturing the population of NRPs as suspension aggregates in medium comprising a y-secretase inhibitor and a ROCK
inhibitor.
inhibitor.
4. The method of claim 1, wherein the culturing of steps (a)-(d) is further defined as adherent 2-dimensional culture.
5. The method of claim 2, wherein the RIM comprises a BMP inhibitor, a TGFr3 inhibitor, and/or IGF-1.
6. The method of claim 5, wherein the RIM further comprises a WNT
inhibitor.
inhibitor.
7. The method of claim 5, wherein RIM is essentially free of or free of CKI-7.
8. The method of claim 2, wherein the RIM is essentially free of activin A.
9. The method of claim 1, wherein the RD1 medium further comprises a TGF13 inhibitor, a Wnt inhibitor, and a MEK inhibitor.
10. The method of claim 9, wherein the RD1 further comprises a Wnt inhibitor.
11. The method of claim 9, wherein the RD1 is essentially free of Wnt inhibitors.
12. The method of claim 11, wherein the RD1 is essentially free or free of CKI-7.
13. The method of claim 1, wherein the RD2 medium comprises a TGF13 inhibitor, a Wnt inhibitor, and a MEK inhibitor.
14. The method of claim 13, wherein the RD2 medium is essentially free of LDN193189.
15. The method of claim 1, wherein the RIVI medium comprises nicotinamide and ascorbic acid.
16. The method of claim 15, wherein the RM medium further comprises FGF and a TGFr3 inhibitor.
17. The method of claim 15 or 16, wherein the RIVI medium further comprises a y-secretase inhibitor.
18. The method of claim 1, further comprising detecting an increase in VSX2 expression of the anterior neuroectoderm cells to determine differentiation potential.
19. The method of claim 1, wherein at least 90% of the cells after culturing in RD2 express PMEL17.
20. The method of claim 1, wherein at least 30% of the cells after culturing in RD2 express VSX2.
21. The method of claim 1, wherein the RPCs express PAX6, MITF, and/or PMEL.
22. The method of claim 1 or 21, wherein the RPCs do not express or have essentially no expression of TRYP1, CRALBP, and/or BEST1.
23. The method of claim 1, wherein at least 70% of the cells after culture in RIVI express PAX6 and CHX10 (VSX2).
24. The method of claim 1, wherein the NRPs express PAX6 and CHX10 (VSX2).
25. The method of claim 1, wherein the NRPs express one or more of the markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL.
26. A pharmaceutical composition comprising the NRPs produced according to claim 1 and a pharmaceutically acceptable carrier.
27. A method for producing a population of photoreceptor precursor cells (PRPs) comprising:
(a) obtaining a starting population of NRPs according to any of claims 1-25;
(b) further culturing the NRPs in photoreceptor precursor induction medium (FDSC) comprising a y-secretase inhibitor and FGF for a period of time sufficient to produce a population of PRPs.
(a) obtaining a starting population of NRPs according to any of claims 1-25;
(b) further culturing the NRPs in photoreceptor precursor induction medium (FDSC) comprising a y-secretase inhibitor and FGF for a period of time sufficient to produce a population of PRPs.
28. The method of claim 27 wherein the FDSC further comprises a TGFr3 inhibitor and WNT inhibitor.
29. The method of claim 27, wherein the culturing of steps (a) and (b) is further defined as adherent 2-dimensional culture.
30. The method of claim 27, further comprising maturing the population of PRPs as suspension aggregates in RIVI medium or a photoreceptor maturation (PM) medium comprising nicotinamide, thereby producing a population of mature PRP
aggregates.
aggregates.
31. The method of claim 30, further comprising cryopreserving the mature PRP
aggregates.
aggregates.
32. The method of claim 30, further comprising dissociating the mature PRP
aggregates into essentially single cells in PM medium.
aggregates into essentially single cells in PM medium.
33. The method of claim 32, further comprising cryopreserving the mature PRPs as single cells.
34. The method of clam 30, wherein the PM medium further comprises a y-secretase inhibitor.
35. The method of claim 27, further comprising purifying the PRPs.
36. The method of any of claims 27-35, wherein purifying comprises selecting cells that are positive for CD171, thereby providing a purified PRP cell population.
37. The method of claim 35, wherein purifying comprises selecting cells that are positive for CD171 and/or SUSD2, thereby providing a purified PRP cell population.
38. The method of claim 35, wherein purifying comprises selecting cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, thereby providing a purified PRP cell population.
39. The method of claim 35, wherein purifying comprises selecting cells that are positive for CD111, CD133, CD230, and/or CD344, thereby providing a purified PRP cell population.
40. The method of claim 35, wherein purifying comprises selecting cells that are positive for CD344, thereby providing a purified PRP cell population.
41. The method of any of claims 35-40, wherein purifying comprises depletion of cells positive for two or more of the markers selected from the group consisting of CD9, CD49f, CD340, podoplanin, CD29, CD63, and CD298.
42. The method of any of claims 35-41, wherein at least 90% of the cells express Class III
0-tubu1in (TUBB3).
0-tubu1in (TUBB3).
43. The method of any of claims 35-42, wherein at least 75% of the cells express Recoverin (RCVRN).
44. The method of any of claims 35-43, wherein at least 50% of the cells express RCVRN.
45. The method of any of claims 35-45, wherein at least 70% of the cells express RCVRN.
46. The method of claim 27, wherein the PRPs express one or more markers selected from the group consisting of 0TX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, RCVRN, TUBB3 and CD171/L1CAM.
47. The method of claim 27, wherein the PRPs do not express or have essentially no expression of TRYP1, CRALBP, BEST1, Ki67, MITF, PMEL17, PAX6, CHX10 and/or Onecutl.
48. The method of claims 35-40, wherein less than 15%, 10%, or 5% of the cells in the purified PRP population express PAX6.
49. A pharmaceutical composition comprising the PRPs produced according to claim 27 and a pharmaceutically acceptable carrier.
50. A method for producing a population of PRPs comprising:
(a) obtaining a starting population of NRPs according to any one of claims 1-24;
(b) culturing the NRPs as aggregates in RIVI medium; and (c) further culturing the NRPs in RM medium or PRP maturation medium (PM) further comprising a cyclin-dependent kinase inhibitor for a period of time sufficient to produce a population of PRPs.
(a) obtaining a starting population of NRPs according to any one of claims 1-24;
(b) culturing the NRPs as aggregates in RIVI medium; and (c) further culturing the NRPs in RM medium or PRP maturation medium (PM) further comprising a cyclin-dependent kinase inhibitor for a period of time sufficient to produce a population of PRPs.
51. The method of claim 50, wherein at least 70% of the cells are positive for PAX6 and CHX10 prior to step (b).
52. The method of claim 50, wherein the NRPs are cultured as aggregates in the presence of a y-secretase inhibitor and a ROCK inhibitor.
53. The method of claim 50, wherein the PM further comprises a y-secretase inhibitor
54. The method of claim 53, wherein the PM further comprises a MEK
inhibitor.
inhibitor.
55. The method of claim 50, further comprising dissociating the PRPs into essentially single cells in PM medium.
56. The method of claim 55, wherein the PRPs are cultured as adherent cells in PM
medium.
medium.
57. The method of claim 55, further comprising enriching for PRPs by selecting for cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, and/or removing cells that are positive for CD9, CD49f, CD340, podoplanin CD29, CD63, and/or CD298.
58. The method of claim 57, wherein at least 90% of the cells are positive for TUBB3 and/or at least 70% of the cells are positive for RCVRN.
59. A pharmaceutical composition comprising the PRPs produced according to claim 50 and a pharmaceutically acceptable carrier.
60. A method for producing a population of optic vesicles (0Vs) comprising:
(a) obtaining a starting population of PRPs according to claim 50; and (b) further culturing the PRPs as suspension aggregates in RIVI medium or PM
medium for a period of time sufficient to produce a population of OVs.
(a) obtaining a starting population of PRPs according to claim 50; and (b) further culturing the PRPs as suspension aggregates in RIVI medium or PM
medium for a period of time sufficient to produce a population of OVs.
61. The method of claim 60, wherein the RM medium or PM medium comprises a y-secretase inhibitor.
62. The method of claim 61, wherein the PM medium further comprises a inhibitor.
63. The method of claim 60, wherein at least 50% of the cells in the population of OVs express VSX2.
64. The method of claim 60, wherein at least 20% of the cells in the population of OVs express RCVRN.
65. The method of claim 60, wherein the OVs express Gamma-synuclein (SNCG), Opsin, RCVRN, and Rhodopsin.
66. A pharmaceutical composition comprising the OVs produced according to claim 60 and a pharmaceutically acceptable carrier.
67. A composition comprising an NRP population, wherein at least 90% of the cells in the NRP population express PAX6, at least 90% of the cells in the NRP population express PMEL17, and/or at least 70% of the cells in the NRP population express VSX2.
68. The composition of claim 67, wherein at least 95% of the cells in the NRP population express PAX6, at least 90% of the cells in the NRP population express PMEL17, and/or at least 75% of the cells in the NRP population express VSX2.
69. The composition of claim 67, wherein at least 95% of the cells in the NRP population express PAX6, at least 90% of the cells in the NRP population express PMEL17, and at least 75% of the cells in the NRP population express VSX2.
70. The composition of claim 71, wherein the cells in the NRP population further express Ki67.
71. A composition comprising a PRP population, wherein at least 90% of the cells in the PRP population express TUBB3, at least 50% of the cells in the PRP cell population express RCVRN, and/or less than 15% of the cells in the PRP population express PAX6.
72. The composition of claim 67, wherein at least 70% of the cells in the PRP population express RCVRN.
73. The composition of claim 67, wherein less than 10% or 5% of the cells in the PRP
population express PAX6.
population express PAX6.
74. The composition of claim 67, wherein at least 90% of the cells in the PRP population express TUBB3, at least 50% of the cells in the PRP cell population express RCVRN, and less than 15% of the cells in the PRP population express PAX6.
75. The composition of claim 67, wherein at least 90% of the cells in the PRP population express TUBB3, at least 70% of the cells in the PRP cell population express RCVRN, and/or less than 5% of the cells in the PRP population express PAX6.
76. The composition of any one of claims 67-75, wherein the PRPs express one or more markers selected from the group consisting of 0TX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, RCVRN, TUBB3 and CD171/L1CAM.
77. The composition of claim 76, wherein the PRPs do not express or have essentially no expression of TRYP1, CRALBP, BEST1, Ki67, MITF, and/or PMEL17.
78. A composition comprising a population of OVs, wherein at least 50% of the cells express VSX2 and/or at least 20% of the cells express RCVRN.
79. The composition of claim 78, wherein at least 65% of the cells express VSX2 and/or at least 30% of the cells express RCVRN.
80. The composition of claim 78, wherein at least 65% of the cells express VSX2 and at least 30% of the cells express RCVRN.
81. A method of treating injury or degeneration of retinal neurons in a subject comprising administering an effective amount of the composition of claim 42 or 71 to an eye of the subj ect.
82. The method of claim 81, wherein the retinal neurons are photoreceptors.
83. A method for providing an enriched population of PRP cells comprising:
a) obtaining a starting cell population comprising PRP cells; and b) enriching said starting cell population for PRP cells by selecting for cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, and/or removing therefrom cells that are positive for CD9, CD49f, CD340, podoplanin, CD29, CD63, and/or CD298, thereby providing a PRP-enriched cell population that is enriched for PRP
cells as compared to the starting cell population.
a) obtaining a starting cell population comprising PRP cells; and b) enriching said starting cell population for PRP cells by selecting for cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and/or PTK7, and/or removing therefrom cells that are positive for CD9, CD49f, CD340, podoplanin, CD29, CD63, and/or CD298, thereby providing a PRP-enriched cell population that is enriched for PRP
cells as compared to the starting cell population.
84. The method of claim 83, further comprising determining a level of enrichment of the PRP cells in said PRP-enriched population.
85. The method of claim 84, wherein the level of enrichment is determined through the use of a cell marker selected from the group consisting of TUBB3, RCVRN, OTX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, and CD171/L1CAM.
86. The method of claim 83, wherein the PRP-enriched cell population is enriched for PRP cells as compared to the starting cell population as determined by RCVRN
sorting.
sorting.
87. The method of claim 86, wherein the PRP-enriched population is at least 50% PRP
cells.
cells.
88. The method of claim 86, wherein the PRP-enriched population is at least 70% PRP
cells.
cells.
89. The method of claim 86, wherein the PRP-enriched population is essentially pure PRP
cells.
cells.
90. The method of claim 83, wherein the PRP cells are human PRP cells.
91. The method of claim 83, wherein the starting cell population is prepared from iPSCs.
92. The method of claim 83, wherein selecting and/or removing is performed by magnetic bead-based sorting or fluorescence-based sorting.
93. The method of claim 83, wherein the method comprises selecting for cells that are positive for CD171, SUSD2, CD111, CD133, CD230, and/or CD344.
94. A method for performing quality control during the production of an NRP
cell product comprising detecting the expression of cell markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL.
cell product comprising detecting the expression of cell markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL.
95. The method of claim 94, wherein at least 70% of the cells are positive for PAX6, CHX10 (VSX2), Ki67, and PMEL.
96. The method of claim 94, wherein at least 90% of the cells are positive for PAX6, CHX10 (VSX2), Ki67, and PMEL.
97. A method for performing quality control during the production of a PRP
cell product comprising detecting the expression of off-target cell markers selected from the group consisting of PAX6, ONECUT1, HNCHF6, CHX10, and Ki67.
cell product comprising detecting the expression of off-target cell markers selected from the group consisting of PAX6, ONECUT1, HNCHF6, CHX10, and Ki67.
98. The method of claim 1, wherein off-target cells are positive for PAX6 and ISL1.
99. The method of claim 97, wherein a PRP cell product passing quality control comprises less than 10% or 5% PAX6-positive cells.
100. The method of claim 97, wherein a PRP cell product passing quality control comprises less than 10% PAX6-positive cells, less than 0.05% Ki67-positive cells, less than 30%
CHX10-positive cells, and/or less than 2% ONECUT1-positive cells.
CHX10-positive cells, and/or less than 2% ONECUT1-positive cells.
101. The method of claim 97, wherein a PRP cell product passing quality control comprises less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15%
CHX10-positive cells, and/or less than 1% ONECUT1-positive cells.
CHX10-positive cells, and/or less than 1% ONECUT1-positive cells.
102. The method of claim 97, wherein a PRP cell product passing quality control comprises less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15%
CHX10-positive cells, and less than 1% ONECUT1-positive cells.
CHX10-positive cells, and less than 1% ONECUT1-positive cells.
103. A method for performing quality control during the production of an OV
cell product comprising detecting the expression of one or more cell markers selected from the group consisting of RCVRN, CHX10, PAX6, and Ki67.
cell product comprising detecting the expression of one or more cell markers selected from the group consisting of RCVRN, CHX10, PAX6, and Ki67.
104. The method of claim 103, wherein the cells markers are RCVRN and CHX10.
105. The method of claim 104, wherein at least 60% of the cells in the OV cell product are positive for RCVRN and at least 30% of the cells in the OV cell product are positive for CHX10.
106. The method of claim 103, wherein the cell markers are PAX6 and Ki67.
107. The method of any one of claims 103-106, further comprising detecting the absence of TYRP 1.
108. The method of claim 107, further comprising detecting the expression of one or more markers selected from the group consisting of MITF, CRALBP, BEST1, OTX2, CRX, BLIMP1, NEUROD1, TUBB3, ONECUT1, and CD171/L1CAM.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862660899P | 2018-04-20 | 2018-04-20 | |
US62/660,899 | 2018-04-20 | ||
PCT/US2019/028557 WO2019204817A1 (en) | 2018-04-20 | 2019-04-22 | Method for differentiation of ocular cells and use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3097428A1 true CA3097428A1 (en) | 2019-10-24 |
Family
ID=66770532
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3097428A Pending CA3097428A1 (en) | 2018-04-20 | 2019-04-22 | Method for differentiation of ocular cells and use thereof |
Country Status (9)
Country | Link |
---|---|
US (1) | US20210238546A1 (en) |
EP (1) | EP3781675A1 (en) |
JP (1) | JP2021521792A (en) |
KR (1) | KR20210005111A (en) |
CN (1) | CN112204134A (en) |
AU (1) | AU2019256723A1 (en) |
CA (1) | CA3097428A1 (en) |
IL (1) | IL277965A (en) |
WO (1) | WO2019204817A1 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2022551478A (en) | 2019-10-09 | 2022-12-09 | ブルーロック セラピューティクス エルピー | Cells with persistent transgene expression |
CA3178949A1 (en) * | 2020-05-29 | 2021-12-02 | Andrew DIAS | Retinal pigmented epithelium and photoreceptor dual cell aggregates and methods of use thereof |
EP4180515A1 (en) * | 2020-08-18 | 2023-05-17 | Tosoh Corporation | Method for inducing differentiation of pluripotent stem cells into ectodermal, mesodermal, and endodermal cells |
CN114146095A (en) * | 2020-09-07 | 2022-03-08 | 是光隽恒(北京)生物科技有限公司 | Composition for repairing tissue damage and preparation method and application thereof |
TW202300642A (en) | 2021-03-25 | 2023-01-01 | 美商藍岩醫療公司 | Methods for obtaining induced pluripotent stem cells |
CN113528441B (en) * | 2021-08-05 | 2022-09-13 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Rapid and efficient clinical-grade pigment epithelial cell induction method, kit and application |
WO2023211857A1 (en) * | 2022-04-25 | 2023-11-02 | Lineage Cell Therapeutics, Inc. | Methods and compositions for treating vision loss |
WO2024085251A1 (en) * | 2022-10-21 | 2024-04-25 | 住友ファーマ株式会社 | Method for evaluating quality of retinal implant |
Family Cites Families (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6416998B1 (en) | 1992-09-02 | 2002-07-09 | Baylor College Of Medicine | Plasmid encoding a modified steroid hormone |
US5843780A (en) | 1995-01-20 | 1998-12-01 | Wisconsin Alumni Research Foundation | Primate embryonic stem cells |
US5763270A (en) | 1995-06-07 | 1998-06-09 | Genemedicine, Inc. | Plasmid for delivery of nucleic acids to cells and methods of use |
JP2001508302A (en) | 1997-01-10 | 2001-06-26 | ライフ テクノロジーズ,インコーポレイテッド | Embryonic stem cell serum replacement |
JP5943533B2 (en) | 2000-05-17 | 2016-07-06 | アステリアス バイオセラピューティクス インコーポレイテッド | Neural progenitor cell population |
US20030211603A1 (en) | 2001-08-14 | 2003-11-13 | Earp David J. | Reprogramming cells for enhanced differentiation capacity using pluripotent stem cells |
WO2004028563A1 (en) | 2002-09-27 | 2004-04-08 | Genexine Inc. | A vaccine enhancing the protective immunity to hepatitis c virus using plasmid dna and recombinant adenovirus |
US7682828B2 (en) | 2003-11-26 | 2010-03-23 | Whitehead Institute For Biomedical Research | Methods for reprogramming somatic cells |
NZ553235A (en) | 2004-09-08 | 2009-11-27 | Wisconsin Alumni Res Found | Culturing human pluripotent stem cells |
EP3147296A1 (en) | 2005-11-14 | 2017-03-29 | Merial, Inc. | Gene therapy for renal failure |
EP4223769A3 (en) | 2005-12-13 | 2023-11-01 | Kyoto University | Nuclear reprogramming factor |
US8278104B2 (en) | 2005-12-13 | 2012-10-02 | Kyoto University | Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2 |
US8129187B2 (en) | 2005-12-13 | 2012-03-06 | Kyoto University | Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2 |
US9683232B2 (en) | 2007-12-10 | 2017-06-20 | Kyoto University | Efficient method for nuclear reprogramming |
ES2587395T3 (en) | 2008-06-04 | 2016-10-24 | Cellular Dynamics International, Inc. | Procedures for the production of IPS cells using a non-viral approach |
CN102239249A (en) | 2008-10-24 | 2011-11-09 | 威斯康星校友研究基金会 | Pluripotent stem cells obtained by non-viral reprogramming |
US8366563B2 (en) | 2009-02-27 | 2013-02-05 | John Kuhlman | Golf alignment device and method |
CN102459575A (en) | 2009-06-05 | 2012-05-16 | 细胞动力国际有限公司 | Reprogramming t cells and hematophietic cells |
US8900871B2 (en) | 2009-08-07 | 2014-12-02 | Kyoto University | Method of producing induced pluripotent stem cells using inhibitors of P53 |
WO2011090221A1 (en) | 2010-01-22 | 2011-07-28 | Kyoto University | Method for improving induced pluripotent stem cell generation efficiency |
US8278620B2 (en) | 2010-05-03 | 2012-10-02 | Thermo Finnigan Llc | Methods for calibration of usable fragmentation energy in mass spectrometry |
AU2014212230B2 (en) | 2013-02-01 | 2019-07-04 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Method for generating retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (iPSCs) |
US10220117B2 (en) * | 2013-10-09 | 2019-03-05 | The Regents Of The University Of California | Methods of mammalian retinal stem cell production and applications |
US10435667B2 (en) * | 2014-01-16 | 2019-10-08 | The Johns Hopkins University | Methods for forming three-dimensional human retinal tissue in vitro |
DK3211071T3 (en) * | 2014-10-24 | 2021-07-26 | Sumitomo Dainippon Pharma Co Ltd | MANUFACTURING PROCEDURE FOR RETINAL TISSUE |
WO2016103269A1 (en) * | 2014-12-23 | 2016-06-30 | Ramot At Tel-Aviv University Ltd. | Populations of neural progenitor cells and methods of producing and using same |
DK3347456T3 (en) * | 2015-09-08 | 2024-02-19 | Us Health | PROCEDURE FOR REPRODUCIBLE DIFFERENTIATION OF CLINICAL-GRADE RETINAL PIGMENT EPITHELIAL CELLS |
DK3347457T3 (en) * | 2015-09-08 | 2022-01-17 | Fujifilm Cellular Dynamics Inc | MACS-based purification of stem cell-derived retinal pigment epithelium |
WO2017091844A1 (en) * | 2015-12-04 | 2017-06-08 | Bond University Ltd | Methods of differentiating retinal cells |
-
2019
- 2019-04-22 KR KR1020207033429A patent/KR20210005111A/en unknown
- 2019-04-22 EP EP19728764.2A patent/EP3781675A1/en active Pending
- 2019-04-22 US US17/049,018 patent/US20210238546A1/en active Pending
- 2019-04-22 CN CN201980035975.9A patent/CN112204134A/en active Pending
- 2019-04-22 JP JP2020557896A patent/JP2021521792A/en active Pending
- 2019-04-22 CA CA3097428A patent/CA3097428A1/en active Pending
- 2019-04-22 WO PCT/US2019/028557 patent/WO2019204817A1/en active Application Filing
- 2019-04-22 AU AU2019256723A patent/AU2019256723A1/en active Pending
-
2020
- 2020-10-12 IL IL277965A patent/IL277965A/en unknown
Also Published As
Publication number | Publication date |
---|---|
IL277965A (en) | 2020-11-30 |
JP2021521792A (en) | 2021-08-30 |
CN112204134A (en) | 2021-01-08 |
AU2019256723A1 (en) | 2020-11-05 |
WO2019204817A1 (en) | 2019-10-24 |
EP3781675A1 (en) | 2021-02-24 |
KR20210005111A (en) | 2021-01-13 |
US20210238546A1 (en) | 2021-08-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220033770A1 (en) | Macs-based purification of stem cell-derived retinal pigment epithelium | |
US20210139847A1 (en) | Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells | |
US20210238546A1 (en) | Method for differentiation of ocular cells and use thereof | |
US20230201267A1 (en) | Retinal pigmented epithelium and photoreceptor dual cell aggregates and methods of use thereof | |
WO2023196577A1 (en) | Methods for production of ipscs |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20240419 |